Biological synthesis of 6-aminocaproic acid from carbohydrate feedstocks

ABSTRACT

Provided herein are methods for the production of difunctional alkanes in microorganisms. Also provided are enzymes and nucleic acids encoding such enzymes, associated with the difunctional alkane production from carbohydrates feedstocks in microorganisms. The invention also provides recombinant microorganisms and metabolic pathways for the production of difunctional alkanes.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/209,917, filed Mar. 11, 2009, which application is herebyincorporated by reference in its entirety.

FIELD OF INVENTION

Aspects of the invention relate to methods for the production ofdifunctional alkanes in microorganisms. In particular, aspects of theinvention describe enzymes, and nucleic acids encoding such enzymes,associated with the difunctional alkane production from carbohydratesfeedstocks in microorganisms. More specifically, aspects of theinvention describe recombinant microorganisms and metabolic pathways forthe production of adipic acid, aminocaproic acid, hexamethylenediamine,6-hydroxyhexanoate and 6-hydroxyhexanamine and 1,6-hexanediol,5-aminopentanol, 5-aminopentanoate, 1,5-pentanediol, glutarate and5-hydroxypentanoate.

BACKGROUND

Crude oil is the number one starting material for the synthesis of keyorganic chemicals and polymers. As oil becomes increasingly scarce andexpensive, biological processing of renewable raw materials in theproduction of chemicals using live microorganisms or their purifiedenzymes becomes increasingly interesting. Biological processing, inparticular, fermentations have been used for centuries to makebeverages. Over the last 50 years, microorganisms have been usedcommercially to make compounds such as antibiotics, vitamins, and aminoacids. However, the use of microorganisms for making industrialchemicals has been much less widespread. It has been realized onlyrecently that microorganisms may be able to provide an economical routeto certain compounds that are difficult or costly to make byconventional chemical means.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a microorganism producing6-aminocaproic acid from lysine, and the microorganism includes at leastone nucleic acid encoding a polypeptide that catalyzes a substrate toproduct conversion such as:

i) lysine to beta-lysine,

ii) beta-lysine to 6-amino-3-oxohexanoic acid,

iii) 6-amino-3-oxohexanoic acid to 6-amino-3-hydroxyhexanoic acid,

iv) 6-amino-3-hydroxyhexanoic acid to 6-aminohex-2-enoic acid, or

v) 6-aminohex-2-enoic acid to 6-aminocaproic acid.

In some embodiments, the nucleic acid molecule is heterologous to therecombinant microorganism.

In other embodiments, the microorganism also includes a nucleic acidencoding a polypeptide that catalyzes a substrate to product conversionsuch as:

i) 6-amino-3-hydroxyhexanoic acid to 6-amino-3-hydroxyhexanoyl-CoA,

ii) 6-amino-3-hydroxyhexanoyl-CoA to 6-aminohex-2-enoyl-CoA, or

iii) 6-aminohex-2-enoyl-CoA to 6-aminohex-2-enoic acid.

In other embodiments, the microorganism also includes a nucleic acidencoding a polypeptide that catalyzes a substrate to product conversionsuch as:

i) 6-amino-3-hydroxyhexanoyl-CoA to 6-aminohexanoyl-CoA or

ii) 6-aminohexanoyl-CoA to 6-aminocaproic acid.

In a second aspect, the invention provides in part a recombinantmicroorganism producing 6-aminocaproic acid from lysine, and therecombinant microorganism includes at least one nucleic acid encoding apolypeptide that catalyzes a substrate to product conversion such as:

i) lysine to beta-lysine,

ii) beta-lysine to 3,6-diaminohexanoyl-CoA,

iii) 3,6-diaminohexanoyl-CoA to 6-aminohex-2-enoyl-CoA,

iv) 6-aminohex-2-enoyl-CoA to 6-aminohex-2-enoic acid, or

v) 6-aminohex-2-enoic acid to 6-aminocaproic acid. The at least onenucleic acid molecule is generally heterologous to the recombinantmicroorganism.

In another aspect, the invention provides a recombinant microorganismproducing 6-aminocaproic acid from lysine, the recombinant microorganismincludes at least one nucleic acid encoding a polypeptide that catalyzesa substrate to product conversion that includes:

i) lysine to beta-lysine,

ii) beta-lysine to 3,6-diaminohexanoyl-CoA,

iii) 3,6-diaminohexanoyl-CoA to 6-aminohex-2-enoyl-CoA,

iv) 6-aminohex-2-enoyl-CoA to 6-aminohexanoyl-CoA, or

v) 6-aminohexanoyl-CoA to 6-aminocaproic acid.

In a further aspect, provided is a recombinant microorganism producing6-aminocaproic acid from lysine, where the recombinant microorganismincludes at least one nucleic acid encoding a polypeptide that catalyzesa substrate to product conversion such as:

i) lysine to beta-lysine,

ii) beta-lysine to 6-aminohex-2-enoic acid, or

iii) 6-aminohex-2-enoic acid to 6-aminocaproic acid.

In certain embodiments, the at least one nucleic acid molecule isheterologous to the recombinant microorganism.

In a further aspect, provided is a recombinant microorganism producing6-aminocaproic acid from lysine, where the recombinant microorganismincludes at least one nucleic acid encoding a polypeptide that catalyzesa substrate to product conversion such as:

i) lysine to 6-amino-2-oxohexanoic acid,

ii) 6-amino-2-oxohexanoic acid to 6-amino-2-hydroxyhexanoic acid,

iii) 6-amino-2-hydroxyhexanoic acid to 6-aminohex-2-enoic acid, or

iv) 6-aminohex-2-enoic acid to 6-aminocaproic acid.

In certain embodiments, the recombinant microorganism also includes atleast one nucleic acid encoding a polypeptide that catalyzes a substrateto product conversion such as:

i) 6-amino-2-hydroxyhexanoic to 6-amino-2-hydroxyhexanoyl-CoA, or

ii) 6-amino-2-hydroxyhexanoyl-CoA to 6-aminohex-2-enoyl-CoA.

In certain embodiments, the recombinant microorganism also includes atleast one nucleic acid encoding a polypeptide that catalyzes a substrateto product conversion such as:

i) 6-amino-2-hydroxyhexanoic to 6-amino-2-hydroxyhexanoyl-CoA,

ii) 6-amino-2-hydroxyhexanoyl-CoA to 6-aminohexanoyl-CoA, or

iii) 6-aminohexanoyl-CoA to 6-aminocaproic acid.

In another aspect, the invention provides a recombinant microorganismproducing 6-aminocaproic acid from L-2,3-dihydrodipicolinate, and therecombinant microorganism includes at least one nucleic acid encoding apolypeptide that catalyzes a substrate to product conversion such as:

i) L-2,3-dihydrodipicolinate to Δ¹-piperideine-2,6-dicarboxylate,

ii) Δ¹-piperideine-2,6-dicarboxylate to Δ¹-piperideine-2-carboxylate,

iii) Δ¹-piperideine-2-carboxylate to L-pipecolate,

iv) L-pipecolate to 6-amino-2-oxohexanoic acid,

v) 6-amino-2-oxohexanoic acid to 6-amino-2-hydroxyhexanoic acid,

vi) 6-amino-2-hydroxyhexanoic acid to 6-aminohex-2-enoic acid, or

vii) 6-aminohex-2-enoic acid to 6-aminocaproic acid.

In some embodiments the at least one nucleic acid molecule isheterologous to the recombinant microorganism.

In another aspect, provided is a recombinant microorganism producing6-aminocaproic acid from L-2,3-dihydrodipicolinate, where therecombinant microorganism includes at least one nucleic acid encoding apolypeptide that catalyzes a substrate to product conversion such as:

i) L-2,3-dihydrodipicolinate to Δ¹-piperideine-2,6-dicarboxylate,

ii) Δ¹-piperideine-2,6-dicarboxylate to Δ¹-piperideine-2-carboxylate,

iii) Δ¹-piperideine-2-carboxylate to L-pipecolate,

iv) L-pipecolate to 6-amino-2-oxohexanoic acid,

v) 6-amino-2-hydroxyhexanoic to 6-amino-2-hydroxyhexanoyl-CoA,

vi) 6-amino-2-hydroxyhexanoyl-CoA to 6-aminohex-2-enoyl-CoA,

vii) 6-aminohex-2-enoyl-CoA to 6-aminohex-2-enoic acid, or

viii) 6-aminohex-2-enoic acid to 6-aminocaproic acid.

In some embodiments the at least one nucleic acid molecule isheterologous to the recombinant microorganism.

In another aspect, provided is a recombinant microorganism producing6-aminocaproic acid from L-2,3-dihydrodipicolinate, the recombinantmicroorganism includes at least one nucleic acid encoding a polypeptidethat catalyzes a substrate to product conversion such as:

i) L-2,3-dihydrodipicolinate to Δ¹-piperideine-2,6-dicarboxylate,

ii) Δ¹-piperideine-2,6-dicarboxylate to Δ¹-piperideine-2-carboxylate,

iii) Δ¹-piperideine-2-carboxylate to L-pipecolate,

iv) L-pipecolate to 6-amino-2-oxohexanoic acid,

v) 6-amino-2-hydroxyhexanoic to 6-amino-2-hydroxyhexanoyl-CoA,

vi) 6-amino-2-hydroxyhexanoyl-CoA to 6-aminohex-2-enoyl-CoA,

vii) 6-aminohex-2-enoyl-CoA to 6-aminohexanoyl-CoA, or

viii) 6-aminohexanoyl-CoA to 6-aminocaproic acid.

In another aspect, provided is a recombinant microorganism producing6-aminocaproic acid from L-2,3-dihydrodipicolinate, the recombinantmicroorganism includes at least one nucleic acid encoding a polypeptidethat catalyzes a substrate to product conversion such as:

i) L-2,3-dihydrodipicolinate to Δ¹-piperideine-2,6-dicarboxylate,

ii) Δ¹-piperideine-2,6-dicarboxylate to Δ¹-piperideine-2-carboxylate,

iii) Δ¹-piperideine-2-carboxylate to 6-amino-2-oxohexanoic acid,

iv) 6-amino-2-oxohexanoic acid to 6-amino-2-hydroxyhexanoic acid,

v) 6-amino-2-hydroxyhexanoic acid to 6-amino-2-hydroxyhexanoyl-CoA,

vi) 6-amino-2-hydroxyhexanoyl-CoA to 6-amino-hex-2-enoyl-CoA,

vii) 6-amino-hex-2-enoyl-CoA to 6-amino-hex-2-enoic acid, or

viii) 6-amino-hex-2-enoic acid to 6-aminocaproic acid.

In another aspect, provided is a recombinant microorganism producing6-aminocaproic acid from L-2,3-dihydrodipicolinate, the recombinantmicroorganism includes at least one nucleic acid encoding a polypeptidethat catalyzes a substrate to product conversion such as:

i) L-2,3-dihydrodipicolinate to Δ¹-piperideine-2,6-dicarboxylate,

ii) Δ¹-piperideine-2,6-dicarboxylate to 2-amino-6-oxoheptanedioic acid,

iii) 2-amino-6-oxoheptanedioic acid to 6-amino-2-oxohexanoic acid,

iv) 6-amino-2-oxohexanoic acid to 6-amino-hex-2-enoic acid, or

v) 6-amino-hex-2-enoic acid to 6-aminocaproic acid.

In another aspect, provided is a recombinant microorganism producing6-aminocaproic acid from L-2,3-dihydrodipicolinate, the recombinantmicroorganism includes at least one nucleic acid encoding a polypeptidethat catalyzes a substrate to product conversion such as:

i) L-2,3-dihydrodipicolinate to 5,6-dihydropyridine-2-carboxylic acid,

ii) 5,6-dihydropyridine-2-carboxylic acid to 6-amino-2-oxohex-3-enoicacid,

iii) 6-amino-2-oxohex-3-enoic acid to 6-amino-2-oxohexanoic acid,

iv) 6-amino-2-oxohexanoic acid to 6-amino-hex-2-enoic acid, or

v) 6-amino-hex-2-enoic acid to 6-aminocaproic acid.

In another aspect, provided is a recombinant microorganism producing6-aminocaproic acid from L-lysine, the recombinant microorganismincludes at least one nucleic acid encoding a polypeptide that catalyzesa substrate to product conversion such as:

i) L-lysine to D-lysine,

ii) D-lysine to D-pipecolate, or

iii) D-pipecolate to 6-aminocaproic acid.

In another aspect, provided is a recombinant microorganism producingadipic acid from L-2,3-dihydrodipicolinate, the recombinantmicroorganism includes at least one nucleic acid encoding a polypeptidethat catalyzes a substrate to product conversion such as:

i) L-2,3-dihydrodipicolinate to Δ¹-piperideine-2,6-dicarboxylate,

ii) Δ¹-piperideine-2,6-dicarboxylate to Δ¹-piperideine-2-carboxylate,

iii) Δ¹-piperideine-2-carboxylate to L-pipecolate,

iv) L-pipecolate to Δ¹-piperideine-6-carboxylate,

v) Δ¹-piperideine-6-carboxylate to 2-aminoadipate-6-semialdehyde,

vi) 2-aminoadipate-6-semialdehyde to 2-aminoadipate, or

vii) 2-aminoadipate to adipic acid.

In another aspect, provided is a recombinant microorganism producingadipic acid from L-2,3-dihydrodipicolinate, the recombinantmicroorganism includes at least one nucleic acid encoding a polypeptidethat catalyzes a substrate to product conversion such as:

i) L-2,3-dihydrodipicolinate to Δ¹-piperideine-2,6-dicarboxylate,

ii) Δ¹-piperideine-2,6-dicarboxylate to Δ¹-piperideine-2-carboxylate,

iii) Δ¹-piperideine-2-carboxylate to L-pipecolate,

iv) L-pipecolate to Δ¹-piperideine-6-carboxylate,

v) Δ¹-piperideine-6-carboxylate to 2-aminoadipate-6-semialdehyde,

vi) 2-aminoadipate-6-semialdehyde to adipate semialdehyde, or

vii) adipate semialdehyde to adipic acid.

In another aspect, provided is a recombinant microorganism producing

1,6-hexanediol from L-2,3-dihydrodipicolinate, the recombinantmicroorganism includes at least one nucleic acid encoding a polypeptidethat catalyzes a substrate to product conversion such as:

i) L-2,3-dihydrodipicolinate to Δ¹-piperideine-2,6-dicarboxylate,

ii) Δ¹-piperideine-2,6-dicarboxylate to Δ¹-piperideine-2-carboxylate,

iii) Δ¹-piperideine-2-carboxylate to L-pipecolate,

iv) L-pipecolate to Δ¹-piperideine-6-carboxylate,

v) Δ¹-piperideine-6-carboxylate to 2-aminoadipate-6-semialdehyde,

vi) 2-aminoadipate-6-semialdehyde to adipate semialdehyde, or

vii) adipate semialdehyde to 1,6-hexanediol.

In another aspect, provided is a recombinant microorganism producing6-hydroxyhexanoate from L-2,3-dihydrodipicolinate, the recombinantmicroorganism includes at least one nucleic acid encoding a polypeptidethat catalyzes a substrate to product conversion such as:

i) L-2,3-dihydrodipicolinate to Δ¹-piperideine-2,6-dicarboxylate,

ii) Δ¹-piperideine-2,6-dicarboxylate to Δ¹-piperideine-2-carboxylate,

iii) Δ¹-piperideine-2-carboxylate to L-pipecolate,

iv) L-pipecolate to Δ¹-piperideine-6-carboxylate,

v) Δ¹-piperideine-6-carboxylate to 2-aminoadipate-6-semialdehyde,

vi) 2-aminoadipate-6-semialdehyde to adipate semialdehyde, or

vii) adipate semialdehyde to 6-hydroxyhexanoate.

In another aspect, provided is a recombinant microorganism producing6-aminocaproic acid from lysine, the recombinant microorganism includesat least one nucleic acid encoding a polypeptide that catalyzes asubstrate to product conversion such as:

i) lysine to 6-amino-2-oxohexanoic acid,

ii) 6-amino-2-oxohexanoic acid to 7-amino-2-oxoheptanoic acid,

iii) 7-amino-2-oxoheptanoic acid to 6-aminohexanal, or

iv) 6-aminohexanal to 6-aminocaproic acid.

In another aspect, provided is a recombinant microorganism producing6-aminocaproic acid from lysine, the recombinant microorganism includesat least one nucleic acid encoding a polypeptide that catalyzes asubstrate to product conversion such as:

i) lysine to 2,7-diaminoheptanoic acid,

ii) 2,7-diaminoheptanoic acid to 7-amino-2-oxoheptanoic acid,

iii) 7-amino-2-oxoheptanoic acid to 6-aminohexanal, or

iv) 6-aminohexanal to 6-aminocaproic acid.

In another aspect, provided is a recombinant microorganism producing6-aminocaproic acid from lysine, the recombinant microorganism includesat least one nucleic acid encoding a polypeptide that catalyzes asubstrate to product conversion such as:

i) lysine to 2,7-diaminoheptanoic acid,

ii) 2,7-diaminoheptanoic acid to 7-amino-2-oxoheptanoic acid,

iii) 7-amino-2-oxoheptanoic acid to 6-aminohexanamide, or

iv) 6-aminohexanamide to 6-aminocaproic acid.

In another aspect, provided is a recombinant microorganism producinghexamethylenediamine from lysine, the recombinant microorganism includesat least one nucleic acid encoding a polypeptide that catalyzes asubstrate to product conversion such as:

i) lysine to 2,7-diaminoheptanoic acid, or

ii) 2,7-diaminoheptanoic acid to hexamethylenediamine.

In certain embodiments, the recombinant microorganisms described hereinare bacterial or yeast cells.

In further embodiments, one or more of the nucleic acids describedherein encodes an enzyme. For example, the enzyme exists in a naturalbiological system or, alternatively, the enzyme does not exist in anatural biological system.

In still further embodiments, the nucleic acids encode a plurality ofenzymes and the plurality of enzymes do not exist together in a naturalbiological system.

Also provided are nucleic acid preparations that encode at least onepolypeptide that catalyzes a substrate to product conversion describedherein. In some embodiments, the nucleic acids include plasmids or othervector molecules.

Also provided are nucleic acid preparations containing one or morenucleic acid molecules that are engineered nucleic acids, and theseengineered nucleic acids have less than 99%, less than 95%, less than90%, less than 80%, less than 70%, or less than 60% identity with anatural nucleic acid. Nucleic acid preparations are provided that encodeone or more proteins, which have less than 99%, less than 95%, less than90%, less than 80%, less than 70%, or less than 60% identity with anatural protein.

In another aspect, provided are engineered metabolic pathways for theproduction of 6-aminocaproic acid in a recombinant microorganism, whichinclude a plurality of polypeptides that catalyze the substrate toproduct conversions described herein. In some embodiments, the pluralityof polypeptides does not exist in a natural biological system.

In another aspect, provided is an engineered metabolic pathway for theproduction of adipic acid in a recombinant microorganism, which includesa plurality of polypeptides that catalyze the substrate to productconversions described herein. In some embodiments, the plurality ofpolypeptides does not exist in a natural biological system.

In a further aspect, provided is an engineered metabolic pathway for theproduction of 1,6-hexanediol in a recombinant microorganism includes aplurality of polypeptides that catalyze one or more of the substrate toproduct conversions described herein. In some embodiments, the pluralityof polypeptides does not exist in a natural biological system.

In another aspect, provided is an engineered metabolic pathway for theproduction of 6-hydroxyhexanoate in a recombinant microorganism thatincludes a plurality of polypeptides that catalyze a substrate toproduct conversion described herein.

In another aspect, provided is an engineered metabolic pathway for theproduction of hexamethylenediamine in a recombinant microorganism thatincludes a plurality of polypeptides that catalyze the substrate toproduct conversion described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention can be more fully understood from the following detaileddescription and the figures which form part of the application

FIG. 1 shows the structure of L-lysine

FIG. 2 represents a flow diagram for the bioproduction of aminocaproicacid from lysine.

FIG. 3A represents a flow diagram for the bioproduction of aminocaproicacid from L-2,3-dihydrodipicolinate.

FIG. 3B represents a flow diagram for the bioproduction of adipic acid,1,6-hexanediol, and 6-hydroxyhexanoate from L-pipecolate.

FIG. 4 represents a flow diagram of C6 difunctional hexanes from lysinevia an N-heterocyclic ring intermediate.

FIG. 5 represents a flow diagram for the bioproduction of adipic acid,6-hydroxyhexanoate, and 1,6-hexanediol from lysine.

FIG. 6 represents a flow diagram for the bioproduction of6-hydroxyhexamine and hexamethylenediamine from lysine.

FIG. 7 represents a flow diagram for the bioproduction of C5difunctional alkanes from lysine.

FIG. 8 represents a flow diagram for the bioproduction of C5difunctional alkanes from L-pipecolate.

FIG. 9 represents a flow diagram for the bioproduction of C6difunctional alkanes from lysine via a carbon extension process.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the following terms and phrases shall have the meaningsset forth below. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood to one ofordinary skill in the art.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise.

The terms “comprise” and “comprising” are used in the inclusive, opensense, meaning that additional elements may be included.

The term “including” is used to mean “including but not limited to”.

“Including” and “including but not limited to” are used interchangeably.

All publications mentioned herein are incorporated herein by reference.The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present invention. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.

Aspects of the invention provide methods and materials for producingorganic aliphatic compounds of interest in a rapid, inexpensive andenvironmentally responsible way. As such, the present invention meets anumber of commercial and industrial needs. The term “organic molecule”refers, for example, to any molecule that is made up predominantly ofcarbon and hydrogen, such as, for example, alkanes. Organic compounds ofinterest, such as difunctional alkanes, diols, dicarboxylic acids, etc.can be used to synthesize plastic, nylons and other products usuallyderived from petroleum and hydrocarbons. Aspects of the invention relateto the synthesis of difunctional n-alkanes with hydrocarbon chainsderived from a hydrocarbon chain C_(n) wherein n is a number of fromabout 1 to about 8, such as from about 2 to about 5, from about 3 toabout 4, or preferably from 5 to 6. In a preferred embodiment, thedifunctional n-alkanes are derived from lysine, lysine precursors and/orlysine degradation compounds.

Aspects of the invention relate to the production of difunctionalalkanes of interest in a microorganism and provide methods for theproduction of difunctional alkanes from a carbohydrate source in amicroorganism. As used herein “difunctional alkanes” refers to alkaneshaving two functional groups. The term “functional group” refers, forexample, to a group of atoms arranged in a way that determines thechemical properties of the group and the molecule to which it isattached. Examples of functional groups include halogen atoms, hydroxylgroups (—OH), carboxylic acid groups (—COOH) and amine groups (—NH₂) andthe like. “Alcohol” refers, for example, to an alkyl moiety in which oneor more of the hydrogen atoms has been replaced by an —OH group. Theterm “primary alcohol” refers, for example to alcohols in which the —OHgroup is bonded to a terminal or chain-ending carbon atom, such as in1-hexanol and the like. The term “secondary alcohol” refers, for exampleto alcohols in which the —OH group is bonded to a carbon atom that isbonded to one hydrogen atom and to two other carbon atoms, such as in2-hexanol and the like. The term “tertiary alcohol” refers, for exampleto alcohols in which the —OH group is bonded to a carbon atom that isbonded to three other carbon atoms. “Amine” refers, for example, to analkyl moiety in which one or more of the hydrogen atoms has beenreplaced by an —NH₂ group. “Carbonyl compound” refers, for example, toan organic compound containing a carbonyl group, C═O, such as, forexample, aldehydes, which have the general formula RCOH; ketones, whichhave the general formula RCOR′; carboxylic acids, which have the generalformula RCOOH; and esters, which have the general formula RCOOR′.

The method incorporates microorganisms capable of producing one of thefollowing C6-difunctional alkanes of interest, particularly, adipicacid, amino caproic acid, hexamethylenediamine (HMD), or6-hydroxyhexanoate. Other difunctional alkanes of interest include5-aminopentanol, 5-aminopentanoate, 1,5-pentanediol, glutarate,5-hydroxypentanoate, cadaverine, etc. Several chemical synthesis routeshave been described, for example, for adipic acid and its intermediatessuch as muconic acid and adipate semialdehyde; for caprolactam, and itsintermediates such as 6-amino caproic acid; for hexane 1,6 diamine orhexanemethylenediamine; but only a few biological routes have beendisclosed for some of these organic chemicals. Therefore, aspects of theinvention provide engineered metabolic routes, methods to producedifunctional alkanes from sustainable feedstock, and materialsassociated therewith, including isolated nucleic acids or engineerednucleic acids, polypeptides or engineered polypeptides, and host cellsor genetically engineered host cells. Carbon sources suitable asstarting materials for such biosynthetic pathways include carbohydratesand synthetic intermediates. Examples of carbohydrates which cells arecapable of metabolizing include sugars, dextroses, triglycerides andfatty acids. Intermediate products from metabolic pathway such aspyruvate, oxaloacetate, and 2-ketoglutatrate can also be used asstarting materials. Aspects of the invention relate to engineeredpolypeptides, and polynucleotides encoding such polypeptides, havingenzymatic activity or an improved activity for a natural or unnaturalsubstrate or having broad substrate specificity (e.g., catalyticpromiscuity such as substrate promiscuity). The terms “polypeptide,”“protein” and peptide,” which are used interchangeably herein, refer toa polymer of amino acids, including, for example, gene products,naturally-occurring proteins, homologs, orthologs, paralogs, fragments,and other equivalents, variants and analogs of the forgoing. The term“polypeptide having enzymatic activity” refers to any polypeptide thatcatalyzes a chemical reaction of other substances without itself beingdestroyed or altered upon completion of the reaction. Typically, apolypeptide having enzymatic activity catalyzes the formation of one ormore products from one or more substrates. In some aspects of theinvention, the catalytic promiscuity properties of some enzymes may becombined with protein engineering and may be exploited in novelmetabolic pathways and biosynthesis applications. In some embodiments,existing enzymes are modified for use in organic biosynthesis. In somepreferred embodiments, the enzymes involved in the production of thedifunctional n-alkanes of interest include but are not limited to 2amino-decarboxylases, 2-ketodecarboxylases, terminal-aminotransferases,2-aminotransferases, 2 amino-aldehyde mutase, alcohol dehydrogenases,aldehyde dehydrogenases, amino-aldehyde dehydrogenases, dehydrogenases,dehydratases, CoA ligases, CoA-S transferases, deaminases andthioestrerases. In some embodiments, the reaction mechanism of thereference enzyme(s) may be altered to catalyze new reactions, to change,expand or improve substrate specificity. One should appreciate that ifthe enzyme structure (e.g. crystal structure) is known, enzymesproperties may be modified by rational redesign (see US patentapplication US20060160138, US20080064610 and US20080287320).Modification or improvement in enzyme properties may arise from theintroduction of modifications into a polypeptide chain that may, ineffect, perturb the structure-function of the enzyme and/or alter itsinteraction with another molecule (e.g., association with a naturalsubstrate versus an unnatural substrate). It is well known in the artthat certain regions of a protein may be critical for enzyme activity,for example amino acids involved in catalysis and substrate bindingdomains, such that small perturbations to these regions will havesignificant effects on enzyme function. Some amino acid residues may beat important positions for maintaining the secondary or tertiarystructure of the enzyme, and thus also produce noticeable changes inenzyme properties when modified. In some embodiments, the potentialpathway components are variants of any of the foregoing. Such variantsmay be produced by random mutagenesis or may be produced by rationaldesign for production of an enzymatic activity having, for example, analtered substrate specificity, increased enzymatic activity, greaterstability, etc. Thus, in some embodiments, the number of modificationsto a reference parent enzyme that produces a variant enzyme having thedesired property may comprise one or more amino acids, 2 or more aminoacids, 5 or more amino acids, 10 or more amino acids, or 20 or moreamino acids, up to 10% of the total number of amino acids, up to 20% ofthe total number of amino acids, up to 30% of the total number of aminoacids, up to 40% of the total number of amino acids making up thereference enzyme, or up to 50% of the total number of amino acids makingup the reference enzyme.

Those skilled in the art will understand that the engineered pathwaysexemplified herein are described in relation to, but are not limited to,species specific genes and proteins and that the invention encompasseshomologs and orthologs of such gene and protein sequences. Homolog andortholog sequences possess a relatively high degree of sequenceidentity/similarity when aligned using methods known in the art. Suchhomologs or orthologs can suitably be obtained by means of anyappropriate cloning strategy known to one skilled in the art. In someembodiments, useful polypeptide sequences have at least 30%, at least45%, at least 60%, at least 75%, at least 85%, or at least 95% identityto the amino acid sequence of the reference enzyme of interest.

Aspects of the invention relate to new microorganisms or “geneticallymodified” microorganisms or host cells that have been engineered topossess new metabolic capabilities or new metabolic pathways. As usedherein the terms “host cell” and “microorganism” are usedinterchangeably. As used herein the term “genetically modified,” withreference to microorganisms, refers to microorganisms having at leastone genetic alteration not normally found in the wild type strain of thereference species. In some embodiments, genetically engineeredmicroorganisms are engineered to express or overexpress at least oneparticular enzyme at critical points in a metabolic pathway, and/or toblock the synthesis of other enzymes, to overcome or circumventmetabolic bottlenecks. The term “metabolic pathway” refers to a seriesof two or more enzymatic reactions in which the product of one enzymaticreaction becomes the substrate for the next enzymatic reaction. At eachstep of a metabolic pathway, intermediate compounds are formed andutilized as substrates for a subsequent step. These compounds may becalled “metabolic intermediates.” The products of each step are alsocalled “metabolites.”

Aspects of the invention provide methods for designing and makingengineered metabolic pathways. In some aspects of the invention,alternative pathways for making a product of interest from one or moreavailable and sustainable substrates may be made in one or more hostcells or microorganisms of interest. One should appreciate that theengineered pathway for making difunctional alkanes of interest mayinvolve multiple enzymes and therefore the flux through the pathway maynot be optimum for the production of the product of interest.Consequently, in some aspects of the invention, the carbon flux isoptimally balanced by modulating the activity level of the pathwayenzymes relative to one another. Examples of such modulation areprovided throughout the application. As used herein the term “carbonflux” refers to the number of feedstock molecules (e.g. glucose) whichproceed down the engineered pathway relative to competitive paths.

A host cell as used herein refers to an in vivo or in vitro eukaryoticcell, a prokaryotic cell or a cell from a multicellular organism (e.g.cell line) cultured as a unicellular entity. A host cell may beprokaryotic (e.g., a bacterial cell) or eukaryotic (e.g., a yeast,mammal or insect cell). For example, host cells may be bacterial cells(e.g., Escherichia coli, Bacillus subtilis, Mycobacterium spp., M.tuberculosis, or other suitable bacterial cells), Archaea (for example,Methanococcus Jannaschii or Methanococcus Maripaludis or other suitablearchaic cells), yeast cells (for example, Saccharomyces species such asS. cerevisiae, S. pombe, Picchia species, Candida species such as C.albicans, or other suitable yeast species). Eukaryotic or prokaryotichost cells can be, or have been, genetically modified (also referred as“recombinant”, “metabolically engineered” or “genetically engineered”)and used as recipients for a nucleic acid, such as an expression vector,that comprises a nucleotide sequence encoding one or more biosyntheticor engineered pathway gene products. Eukaryotic and prokaryotic hostcells also denote the progeny of the original cell which has beengenetically engineered by the nucleic acid. In some embodiments, a hostcell may be selected for its metabolic properties. For example, if aselection or screen is related to a particular metabolic pathway, it maybe helpful to use a host cell that has a related pathway. Such a hostcell may have certain physiological adaptations that allow it to processor import or export one or more intermediates or products of thepathway. However, in other embodiments, a host cell that expresses noenzymes associated with a particular pathway of interest may be selectedin order to be able to identify all of the components required for thatpathway using appropriate sets of genetic elements and not relying onthe host cell to provide one or more missing steps.

According to aspects of the invention, aerobic or anaerobicmicroorganisms are metabolically engineered. As used herein, ananaerobic organism is any organism that does not require oxygen forgrowth (i.e. anaerobic conditions), such as certain bacterial cells.Advantageously, the bacterial cell can be an E. coli, C. glutanicum, B.flavum or B. lactofermentum cell; these strains are currently beingemployed industrially to make amino compounds using bacterialfermentation processes. For example, C. glutanicum has been usedextensively for amino acid production (e.g. L-glutamate, L-lysine, seeEggleging L et al., 2005, Handbook for Corynebacterium glutanicum. BocaRaton, USA: CRC Press).

The metabolically engineered cell of the invention is made bytransforming a host cell with at least one nucleotide sequence encodingenzymes involved in the engineered metabolic pathways. As used hereinthe terms “nucleotide sequence”, “nucleic acid sequence” and “geneticconstruct” are used interchangeably and mean a polymer of RNA or DNA,single- or double-stranded, optionally containing synthetic, non-naturalor altered nucleotide bases. A nucleotide sequence may comprise one ormore segments of cDNA, genomic DNA, synthetic DNA, or RNA. In apreferred embodiment, the nucleotide sequence is codon-optimized toreflect the typical codon usage of the host cell without altering thepolypeptide encoded by the nucleotide sequence. In certain embodiments,the term “codon optimization” or “codon-optimized” refers to modifyingthe codon content of a nucleic acid sequence without modifying thesequence of the polypeptide encoded by the nucleic acid to enhanceexpression in a particular host cell. In certain embodiments, the termis meant to encompass modifying the codon content of a nucleic acidsequence as a means to control the level of expression of a polypeptide(e.g., to either increase or decrease the level of expression).Accordingly, aspects of the invention include nucleic acid sequencesencoding the enzymes involved in the engineered metabolic pathways. Insome embodiments, a metabolically engineered cell may express one ormore polypeptides having an enzymatic activity necessary to perform thesteps described throughout the description. For example, a particularcell comprises one, two, three, four, five or more than five nucleicacid sequences with each one encoding the polypeptide(s) necessary toperform the conversion of lysine, lysine metabolite precursor and/orlysine degradation metabolites into difunctional alkane(s).Alternatively, a single nucleic acid molecule can encode one, or morethan one, polypeptide. For example, a single nucleic acid molecule cancontain nucleic acid sequences that encode two, three, four or even fivedifferent polypeptides. Nucleic acid sequences useful for the inventiondescribed herein may be obtained from a variety of sources such as, forexample, amplification of cDNA sequence, DNA libraries, de novosynthesis, excision of genomic segments, etc. The sequences obtainedfrom such sources may then be modified using standard molecular biologyand/or recombinant DNA technology to produce nucleic acid sequenceshaving the desired modifications. Exemplary methods for modification ofnucleic acid sequences include for example, site directed mutagenesis,PCR mutagenesis, deletion, insertion, or substitution, or swappingportions of the sequence using restriction enzymes, optionally incombination with ligation, homologous recombination, site specificrecombination or various combination thereof. In other embodiments, thenucleic acid sequence may be a synthetic nucleic acid sequence.Synthetic polynucleotide sequences may be produce using a variety ofmethods described in U.S. Pat. No. 7,323,320, and in copendingapplication having Ser. No. 11/804,996 and in U.S. Patent PublicationNos. 2006/0160138, 2007/0269870, 2008/0064610, and 2008/0287320.

Methods of transformation for bacteria, plant, and animal cells are wellknown in the art. Common bacterial transformation methods includeelectroporation and chemical modification.

In some embodiments, a genetically modified host cell is geneticallymodified such that it produces, when cultured in vitro in a suitablemedium, the product of interest or an intermediate at a level of atleast 0.1 g/l, at least 1 g/l, at least 10 g/l, at least 50 g/l, atleast 100 g/l or at least 150 g/l. One should appreciate that the levelof the metabolite of interest or its metabolic intermediates produced bya genetically modified host cell can be controlled in various ways. Insome embodiment, the level of expression is controlled by the number ofcopies of the nucleic acid sequences encoding one or more enzymesinvolved in the engineered pathway that are contained in the host cell(e.g. high copy expression vector versus medium or low copy expressionvectors). Preferably, the nucleic acid sequences are introduced into thecell using a vector. Low copy expression vectors generally provide fewerthan 20 vector copies per cell (e.g. from 1 to about 5, from 5 to about10, from 10 to about 15, from 15 to about 20 copies of the expressionvector per cell). Suitable low copy expression vectors for prokaryoticcells (e.g. E. Coli) include, but are not limited to pAYC184,pBeloBac11, pBR332, pBAD33, pBBR1MCS and its derivatives, pSC101,SuperCos (cosmid) and pWE15 (cosmid). Medium copy number expressionvectors generally provide from about 20 to about 50 expression vectorscopies per cell or form about 20 to 80 expression vectors copies percell. Suitable medium copy expression vectors for prokaryotic cells(e.g. E. Coli) include, but are not limited to, pTrc99A, pBAD24 andvectors containing a ColE1 origin of replication and its derivatives.High copy number expression vectors generally provide from about 80 toabout 200 or more expression vector copies per cell. Suitable high copyexpression vectors for prokaryotic cells (e.g. E. Coli) include, but arenot limited to, pUC, PCV1, pBluescript, pGEM and pTZ vectors.

Aspects of the invention provide expression cassettes comprising anucleic acid or a subsequence thereof encoding a polypeptide involved inthe engineered pathway. In some embodiments, the expression cassette cancomprise the nucleic acid operably linked to control sequences, such asa transcriptional element (e.g. promoter) and to a terminator. As usedherein, the term “cassette” refers to a nucleotide sequence capable ofexpressing a particular gene if the gene is inserted so as to beoperably linked to one or more regulatory sequences present in thenucleotide sequence. Thus, for example, the expression cassette maycomprise a heterologous gene which is desired to be expressed in thehost cell. In some embodiments, one or more expression cassettes may beintroduced into a vector by known recombinant techniques. A promoter isa sequence of nucleotides that initiates and controls the transcriptionof a desired nucleic acid sequence by an RNA polymerase enzyme. In someembodiments, the promoter may be inducible. In other embodiment,promoters may be constitutive. Non limiting examples of suitablepromoters for the use in prokaryotic host cells include a bacteriophageT7 RNA polymerase promoter, a trp promoter, a lac operon promoter andthe like. Non limiting examples of suitable strong promoters for the usein prokaryotic cells include lacUV5 promoter, T5, T7, Trc, Tac and thelike. Non limiting examples of suitable promoters for use in eukaryoticcells include a CMV immediate early promoter, a SV40 early or latepromoter, a HSV thymidine kinase promoter and the like. Terminationcontrol regions may also be derived from various genes native to thepreferred host.

In some embodiments, a first enzyme of the engineered pathway may beunder the control of a first promoter and the second enzyme of theengineered pathway may be under the control of a second promoter,wherein the first and the second promoter have different strengths. Forexample, the first promoter may be stronger than the second promoter orthe second promoter may be stronger than the first promoter.Consequently, the level of a first enzyme may be increased relative tothe level of a second enzyme in the engineered pathway by increasing thenumber of copies of the first enzyme and/or by increasing the promoterstrength to which the first enzyme is operably linked to relative to thepromoter strength to which the second enzyme is operably linked to. Insome other embodiments, the plurality of enzymes of the engineeredpathway may be under the control of the same promoter. In otherembodiments, altering the ribosomal binding site affects relativetranslation and expression of different enzymes in the pathway. Alteringthe ribosomal binding site can be used alone to control relativeexpression of enzymes in the pathway, or it can be used in concert withthe aforementioned promoter modifications and codon optimization thatalso affects gene expression levels.

In an exemplary embodiment, expression of the potential pathway enzymesmay be dependent upon the presence of a substrate that the pathwayenzyme will act on in the reaction mixture. For example, expression ofan enzyme that catalyzes conversion of A to B may be induced in thepresence of A in the media. Expression of such pathway enzymes may beinduced either by adding the compound that causes induction or by thenatural build-up of the compound during the process of the biosyntheticpathway (e.g., the inducer may be an intermediate produced during thebiosynthetic process to yield a desired product).

One should appreciate that the designation of the enzymes are governedby the specific reaction catalyzed by them as is depicted in FIGS. 2-9.It is possible for a single enzyme to catalyze two reactions that arechemically identical but are assigned to different pathways on the basisof the respective substrate. This may be associated with differentenzyme classification numbers (e.g. EC numbers). In some instance,enzymes have not been yet allocated an EC number, which is why referenceis only made for definition of the relevant enzymatic reaction.

In some embodiments, computer-implemented design techniques may be usedto generate alternative pathways for generating an organic compound ofinterest. In some embodiments, the databases contain genomic informationand their link may be utilized for designing novel metabolic pathways.Examples of database are MetaCyc (a database of metabolic pathways andenzymes), the University of Minnesota biocatalysis/biodegradationdatabase (a database of microbial catalytic reactions and biodegradationpathways for organic chemical compounds), LGAND (a composite databasethat provides information about metabolites and other chemicalcompounds, substrate-product relations representing metabolic and otherreactions and information about enzyme molecules). A database of pathwaycomponents may also contain components of predicted, putative, orunknown functions. It may also contain pseudo-components of definedfunction that may have an undefined composition. In some embodiments, aprogram may design combinations of regulatory and/or functional elementsthat are in the public domain (e.g., that are not covered by patentrights and/or are not subject to a licensing fee). Databases of freelyavailable genetic elements may be generated and/or used as a source ofnucleic acid sequences that can be combined to produce alternativepathways. Alternative pathways containing different combinations ofknown functional and/or regulatory elements (e.g., from differentspecies) may be designed, assembled, and/or tested. Libraries includingvariations in enzymatic element regions may be used to ascertain therelative effects of different types of enzymes or of different variantsof the same enzyme. Libraries including variations in regulatory elementregions may be used to ascertain the optimal expression level orregulatory control among a set of genes. In some embodiments, thefunctional properties of different engineered pathways may be tested invivo by transforming host cells or organisms with the appropriateassembled nucleic acids, and assaying the properties of the engineeredorganisms. In some embodiments, the functional properties of differentengineered pathways may be tested in vitro by isolating componentsexpressed from assembled nucleic acids and testing the appropriatecombinations of components in an in vitro system.

I. Engineered Pathways for the Production of C6 Difunctional Alkanes

Aspects of the invention relate to design and assembly of engineeredpathways for the production of C6 difunctional alkanes of interest.Particularly, aspects of the invention relate to the production ofadipic acid, amino caproic acid (a stable precursor of caprolactamacid), hexamethylene diamine, 6-amino hexanol, 1,6-hexanediol and6-hydroxyhexanoate.

A. Background on C6 Difunctional Hexane Molecules

1. Overview on Adipic Acid:

In 2005, global demand for adipic acid was 2.7 million metric tons.Historically the demand for adipic acid has grown 2% per year and a 2-3%increase is expected through the year 2009. Adipic acid consistentlyranks as one of the top fifty chemicals produced in the US. Nearly 90%of domestic adipic acid is used to produce nylon-6,6. Other uses ofadipic acid include production of lubricants resins, polyester polyolsand plasticizers, and food acidulant.

There are three major commercial production processes: cyclohexaneprocess, cyclohexanol process, butadiene carbonylation process. Thedominant industrial process for synthesizing adipic acid employs initialair oxidation of cyclohexane to yield a mixture of cyclohexanone(ketone) and cyclohexanol (alcohol), which is designated KA (see forexample U.S. Pat. No. 5,221,800). Hydrogenation of phenol to yield KA isalso used commercially, although this process accounts for just 2% ofall adipic acid production. KA produced via both methods is oxidizedwith nitric acid to produce adipic acid. Reduced nitrogen oxidesincluding NO₂, NO, and N₂O are produced as by-products and are recycledback to nitric acid at varying levels. It is becoming increasingly moreinteresting to industry and beneficial to the environment to engineernon-synthetic, biological routes to adipic acid. A number ofmicrobiological routes have been described. Wild-type and mutantorganisms have been shown to convert renewable feedstocks such asglucose and other hydrocarbons to adipic acid (see for exampleWO9507996, and U.S. Pat. No. 5,272,073, U.S. Pat. No. 5,487,987 and U.S.Pat. No. 5,616,496). Similarly, organisms possessing nitrilase activityhave been shown to convert nitriles to carboxylic acids including adipicacid (see for example U.S. Pat. No. 5,629,190). Additionally, wild-typeorganisms have been used to convert cyclohexane and cyclohexanol andother alcohols to adipic acid (see for example U.S. Pat. No. 6,794,165;and US Patent Applications No 2003087403 and 20020127666). For example,in one enzymatic pathway, cyclohexanol is converted in adipic acid, theenzymatic pathway comprising genes isolated from an Acinetobacterencoding hydroxylacylCoA dehydrogenase; enoylCoA hydratase, acylCoAdehydrogenase, ubiquinone oxidoreductase, monoxygenase, aldehydedehydrogenase. Another enzymatic pathway for the conversion ofcyclohexanol to adipic acid has been suggested as including theintermediates cyclohexanol, cyclohexanone, 2-hydroxycyclohexanone,ε-caprolactone, 6-hydroxycaproic acid. Some specific enzyme activitiesin this pathway have been demonstrated, including cyclohexanoldehydrogenase, NADPH-linked cyclohexanone oxygenase, ε-caprolactonehydrolase, and NAD (NADP)-linked 6-hydroxycaproic acid dehydrogenase(Tanaka et al., Hakko Kogaku Kaishi (1977), 55(2), 62-7). An alternateenzymatic pathway has been postulated to comprise cyclohexanol,cyclohexanone, 1-oxa-2-oxocycloheptane, 6-hydroxyhexanoate,6-oxohexanoate and adipate (Donoghue et al., Eur. J. Biochem., 1975,60(1), 1-7).

2. Caprolactam and 6-aminocaproic Acid Overview

Aminocaproic acid (or ε-aminocaproic acid, or IUPAC name 6-aminohexanoicacid) is a possible intermediate for the production of caprolactam.Caprolactam is primarily used in the manufacture of synthetic fibers,especially nylon 6 that is also used in bristle brushes, textilestiffeners, film coatings, synthetic leather, plastics, plasticizers,vehicles, cross linking for polyurethanes, and in the synthesis oflysine. About 2.5 billion tons of nylon 6 is produced annually on aworldwide basis. The production of nylon 6 is accomplished by the ringopening polymerization of the monomer ε-caprolactam. The startingchemical compound for the production of ε-caprolactam is benzene whichis converted to either cyclohexane or phenol and either chemical isconverted via cyclohexanone to cyclohexanone oxime and then thisintermediate is heated in sulfuric acid.

3. Other C6 Difunctional Alkanes

Hexamethylene diamine is mostly used for the production of Nylon 6,6,Nylon 6,10, Nylon 6,66. Nylon 6,6 and nylon 6,10 can be made intovarious kinds of nylon resins and nylon fiber. 6-hydroxyhexanoate (6HH)is a 6-carbon hydroxyalkanoate that can be circularized to caprolactoneor directly polymerized to make polyester plastics (polyhydroxyalkanoatePHA). 1,6-hexanediol is a valuable intermediate for the chemicalindustry. It has applications in a variety of polymer syntheses such asthe production of polyesters for polyurethane elastomers and polymericplasticizers and is also used in gasoline refining.

The problem to be solved therefore is to provide a synthesis route fordifunctional alkanes which not only avoids reliance on environmentallysensitive starting materials such as petroleum but also makes efficientuse of non-petrochemical inexpensive, renewable resources. It wouldfurther be desirable to provide a synthesis route for difunctionalalkanes which avoids the need for significant energy inputs, optimizescarbon flux and minimizes the formation of toxic by-products.

B. Engineered Pathways for the Production of 6-Aminocaproic Acid and itsIntermediates from Lysine

Lysine (Lys or K) is a 1,2,6-trifunctional hexane with the chemicalformula NH₂(CH₂)₄CHNH₂COOH (FIG. 1). L-lysine is an important economicproduct obtained principally by industrial-scale fermentation utilizingthe Gram positive Corynebacterium glutamicum, Brevibacterium flavum andBrevibacterium lactofermentum. A considerable amount is known regardingthe biochemical pathway for L-lysine synthesis in Corynebacteriumspecies (see for example WO04013341 and WO01665730). In plants andmicroorganisms, lysine is synthesized from aspartic acid, which is firstconverted to β-aspartyl-semialdehyde, cyclizated into dihydropicolinate,which is then reduced to Δ¹-piperidine-2,6-dicarboxylate. Ring-openingof this heterocycle gives a series of derivatives of pimelic acid, andultimately leads to lysine. Enzymes involves in the lysine biosynthesisinclude (Lehninger, A. L., D. L. Nelson, and M. M. Cox. 2000. LehningerPrinciples of Biochemistry, 3rd ed. New York: Worth Publishing):aspartokinase, β-aspartate semialdehyde dehydrogenase, dihydropicolinatesynthase, Δ1-piperidine-2,6-dicarboxylate dehydrogenase,N-succinyl-2-amino-6-ketopimelate synthase, succinyl diaminopimelateaminotransferase, succinyl diaminopimelate desuccinylase,diaminopimelate epimerase, diaminopimelate decarboxylase. In mammals,lysine is metabolized to give acetyl-CoA, via an initial transaminationwith α-ketoglutarate. The bacterial degradation of lysine yields tocadaverine by decarboxylation.

One skilled in the art will appreciate that lysine is an ideal precursorof difunctional alkanes as it can be produced in high yield and itcontains an unbroken 6-carbon-chain with functionalized ends. Aspects ofthe invention provide potential pathways for the bioproduction of C6difunctional alkanes. Detailed explanations of the relevant metabolicpathways are given hereinafter.

Aspects of the invention provide several metabolic pathways that can beused to produce organic compounds such as aminocaproic acid and itsintermediates from lysine. These pathways are shown in FIGS. 2-3.Accordingly, aspects of the invention provide a recombinantmicroorganism having an engineered aminocaproic acid biosyntheticpathway. In some embodiments, L-lysine production involves the use ofmolecular biology techniques to augment L-lysine production.Accordingly, in some embodiments, recombinant microorganisms have atleast one gene that is expressed at a level lower or higher than thatexpressed prior to manipulation of the microorganism or in a comparablemicroorganism which has not been manipulated. Genes are selected fromthe group of genes which play a key role in the biosynthesis of lysinesuch as aspartokinase, aspartate semialdehyde dehydrogenase,dihydrodipicolinate synthase, dihydrodipicolinate reductase,tetrahydrodipicolinate succinylase, succinyl-amino-ketopimelatetransaminase, succinyl-diamino-pimelate desuccinylase, diaminopimelateepimerase, diaminopimelate dehydrogenase, arginyl-tRNA synthetase,diaminopimelate decarboxylase, pyruvate carboxylase, phosphoenolpyruvatecarboxylase, glucose-6-phosphate dehydrogenase, transketolase,transaldolase, phosphogluconolactonase, fructose 1,6-biphosphatase,homoserine dehydrogenase, phophoenolpyruvate carboxykinase, succinyl-CoAsynthetase, methyl-malonyl-CoA mutase.

As described in FIG. 2, 6-aminohex-2-enoic and/or 6-aminohex-2-enoyl-CoAare intermediates to several of the aminocaproic acid biosyntheticpathway. Accordingly, other aspects of the invention provide arecombinant microorganism having an engineered 6-aminohex-2-enoic acidor 6-aminohex-2-enoyl-CoA biosynthetic pathway. In some embodiments, the6-aminohex-2-enoic acid biosynthetic pathway is the same as theaminocaproic biosynthetic pathway with omission of the last enzymaticstep. In a preferred embodiment, the engineered microorganisms are usedfor the commercial production of aminocaproic acid or its intermediates.One skilled in the art will appreciate that aminocaproic acidNH₂(CH₂)₅COOH is a derivative and analogue of the amino acid lysineNH₂(CH₂)₄CHNH₂COOH and lysine is therefore an ideal biological precursorof the aminocaproic acid. Aspects of the invention relate to a processfor aminocaproic acid production and applying at least one enzymaticstep A, B, B′ and/or C as illustrated in the pathway shown in FIG. 2.Accordingly, aspects of the invention provide a recombinant host cell ormicroorganism comprising at least one nucleic acid molecule encoding atleast one polypeptide that catalyzes a substrate to product conversionas illustrated in FIG. 2.

Aspects of the invention relate to different possible pathways toaminocaproic acid:

1) Pathway I comprising enzymatic steps A1, B2, B3, B4, and A5;

2) Pathway II comprising enzymatic steps A1, A2, A3, A4 and A5;

3) Pathway III comprising enzymatic steps A1, A2, A3, A6, A7, B5 and B6;

4) Pathway IV comprising enzymatic steps A1, A2, A3, A6, A7, B4 and A5;

5) Pathway V comprising enzymatic steps A1, B2, B3, B5 and B6;

6) Pathway VI comprising enzymatic steps A1, B′ and A5;

7) Pathway VII comprising enzymatic steps C1, C2, C3 and A5;

8) Pathway VIII comprising enzymatic steps C1, C2, C4, C5, B4 and A5;and

9) Pathway IX comprising enzymatic steps C1, C2, C4, C5, B5 and B6.

a) Conversion of Lysine to 3,6-Diaminohexanoic Acid

In some embodiments, the aminocaproic biosynthetic pathway begins withthe conversion of lysine to 3,6-diaminohexanoic acid by action of anaminomutase as shown in the substrate to product conversion step A1 inFIG. 2 (Pathways I through VI). In a preferred embodiment, the lysineaminomutase is a lysine 2,3-aminomutase (KAM or LAM) (EC 5.4.3.2) whichfacilitates the conversion of the amino acid lysine to3,6-diaminohexanoic acid or beta-lysine as shown below:

Lysine 2,3 aminomutase uses Pyridoxal phosphate, Zinc and a 4 Iron-4Sulfur cluster as cofactors and a 5′-deoxyadenosyl radical formed in anS-Adenosyl methionine (SAM) activated radical reaction pathway. Theskilled person will appreciate that polypeptides having an L-lysine2,3-aminomutase activity may be isolated from a variety of sources.Examples of L-lysine 2,3-aminomutase, include but are not limited, to EC5.4.3.2. (encoded by kamA in Fusobacterium nucleatum, Clostridiumsubterminale or Bacillus subtilis), EC 5.4.3.-(encoded by yjeK inEscherichia Coli K1), EC 5.4.3.7 (leucine 2,3-aminomutase fromAndrographis paniculata, Candida utilis, Clostridium lentoputrescens,Clostridium sporogenes, Rattus norvegicus), and EC 5.4.3.6 (Tyrosine2,3-aminomutase).b) Conversion of 3,6-Diaminohexanoic Acid to 6-Amino-3-Oxohexanoic Acid

According to the pathways II, III and IV, 3,6-diaminohexanoic acid (a1)is first converted into 6-amino-3-oxohexanoic acid (b2, step A2).Although there are no reported enzymes that catalyze the substrate toproduct conversion of a1 to a2, the substrate shows some similarity tothose utilized in Table 1 and may be converted by action of an enzymehaving an aminotransferase (or dehydrogenase) activity as listed inTable 1. As meant herein, enzymes having an aminotransferase ordehydrogenase activity are understood to be enzymes that catalyze thetransfer of a 2-amino group from a lysine molecule to a recipientmolecule, leaving behind a β-ketoacid (or 3-oxoacid) molecule containinga primary amino group. The skilled person will appreciate thatpolypeptides useful for converting 3,6-diaminohexanoic acid into6-amino-3-oxohexanoic acid are exemplified but not limited to theenzymes listed in Table 1 and may be isolated from a variety of sources.

TABLE 1 Desired substrate and product EC number Name Gene name(organism) Protein accession number

2.6.1.19 4-aminobutanoate: 2-oxoglutarate aminotransferase GabT from E.Coli puuE from E. Coli UGA1 from S. cerevisiae ABAT from Homo sapiensGabT from Pseudomonas fluorescens

2.6.1.65 N6-acetyl-β-lysine aminotransferase Pseudomonas sp.

1.4.1.11 L-erythro-3,5- diaminohexanoate dehydrogenase kdd fromFusobacterium nucleatum

c) Conversion of 6-Amino-3-Oxohexanoic Acid into6-Amino-3-Hydroxyhexanoic Acid

In some embodiments, the resulting product 6-amino-3-oxohexanoic acid(a2) of enzymatic step A2, is further converted into6-amino-3-hydroxyhexanoic acid (a3) by action of a reductase enzyme(enzymatic step A3, pathways II, III, and IV). Because of thereversibility of most reactions catalyzed by oxido-reductases, enzymaticstep A3 may be catalyzed by a dehydrogenase enzyme. As used herein, thereductase/dehydrogenase enzyme catalyses the carbonyl reduction of theβ-ketones to its corresponding hydroxyl-derivative (secondary alcohol).In some cases, the oxidizing equivalent is supplied in the form of anoxidized nicotinamide cofactor, NAD(+) or NADP(+). Non-limiting examplesof reductase or dehydrogenase enzymes useful in the present inventionare listed in Table 2 and may be available from different sources.Although there are no reported enzymes that catalyze the substrate toproduct conversion of a2 to a3, the substrate shows some similarity tothose utilized in Table 2 and may be converted by action of an enzymehaving a reductase or dehydrogenase activity as listed in Table 2.Preferably, the reductase enzyme is L-carnitine dehydrogenase,3-hydroxypropionate dehydrogenase, (S)-carnitine 3-dehydrogenase,Hydroxyacid-oxoacid transhydrogenase, malonate semialdehyde reductase(NADPH) or 3-oxo-acyl-CoA reductase.

TABLE 2 Desired substrate product reaction EC number Name Gene name(organism) Protein accession number

EC 1.1.1.108 L-camitine dehydrogenase PP0302 from Pseudomonas putida

EC 1.1.1.59 3-hydroxypropiona tedehydrogenase

EC.1.1.1.35 enoyl-CoA hydratase fadJ and fadB from E. Coli

EC 1.1.1.36 phaB (Rhodobacter sphaeroides), PhbB (Zoogloea ramigera)UniProt:Q3IZW0

EC 1.1.1.100

EC 1.1.1.178 HADH2 (Homo sapiens); fadB2x (Pseudomonas putida)

EC 1.1.1.211 HADHA (Homo sapiens); Hadha (rat) P40939; Q64428

EC 1.1.1.212

EC 1.1.1.254 (S)-carnitine 3- dehydrogenase

EC 1.1.1.259

EC 1.1.99.24 Hydroxyacid- oxoacid transhydrogenase

EC 1.1.99.26 3-hydroxycyclo- hexanone dehydrogenase

EC 1.1.1.- malonate semialdehyde reductase (NADPH) (Metallosphaerasedula) malonyl CoA reductase (Chloroflexus aurantiacus)

EC 1.1.1.- 3-oxo-acyl-CoA reductase (Rattus norvegicus)

d) Conversion of 6-Amino-3-Hydroxyhexanoic Acid into(E)-6-Aminohex-2-Enoic Acid

In enzymatic step A4 (pathway II, FIG. 2), 6-amino-3-hydroxyhexanoicacid (a3) is converted into (E)-6-aminohex-2-enoic acid (a4) by adehydratase or hydro-lyase which cleaves carbon-oxygen bonds byelimination of water. Although there are no enzymes reported to catalyzethe enzymatic reaction A4, the substrate (E)-6-aminohex-2-enoic acid issimilar to those utilized by the reductase enzymes listed in Table 3.Preferably, the reductase enzyme is a trans-L-3-hydroxyprolinedehydratase, a dimethyl-maleate hydratase, an L-carnitine dehydratase, afumarate dehydratase, or a 2-oxohept-3-endioate hydroxylation. Oneskilled in the art will appreciate that polypeptides having a reductaseactivity include, but are not limited to the ones listed below and maybe isolated from a variety of sources.

TABLE 3 Desired substrate and product reaction EC number Name Gene name(organism) Protein accession number

EC 4.2.1.17 enoyl-CoA hydratase ((3S)- 3-hydroxyacyl- CoA hydro- lyase)gene: MaoC (E. Coli), PhaJ1 (Pseudomonas aeruginosa), perMFE (Rattusnorvegicus), MFE-2 (Homo sapiens) protein accession number: Q64428(Rattus norvegicus); Q95KZ6 (Bos Taurus)

EC 4.2.1.18 methylglutaconyl- CoA hydratase ((S)-3-hydroxy-3-methylglutaryl- CoA hydro- lyase) AUH (Homo sapiens), Proteinaccession number: Q3HW12 (Acinetobacter sp.); Q13825 (Homo sapiens)

EC 4.2.1.55 Crotonase (3- hydroxybutyryl- CoA dehydratase) Gene:crotonyl-CoA hydratase [(S)-3- hydroxybutyryl- CoA-forming](Metallosphaera sedula) Crotonase: crt1 (Clostridium kluyveri) enoyl-CoAhydratase: ech (Pseudomonas putida) Crotonase: crt (Clostridiumacetobutylicum) Protein accession number: P52046 (Clostridiumacetobutylicum)

EC 4.2.1.58 Crotonoyl-[acyl- carrier-protein] hydratase fatty acidsynthase: FASN (Homo sapiens); β-hydroxyacyl- ACP dehydrase[multifunctional]: fabA (Escherichia coli K12); (β- hydroxyacyl- ACPdehydratase: fabZ (Escherichia coli K12)

EC 4.2.1.59 β-hydroxyacyl- ACP dehydratase fabZ (Escherichia coli K12);fabA (Escherichia coli K12). UniProt accession number: P0A6Q3 (E. Coli),P45159 (Haemophilus influenzae).

EC 4.2.1.74 Long-chain- enoyl-CoA hydratase HADHA (Homo sapiens); Hadha(Rattus norvegicus) UniProt P40939 (Homo sapiens); UniProtQ64428 (Rattusnorvegicus)

EC 4.2.1.77 Trans-L-3- hydroxyproline dehydratase Rattus norvegicus

EC 4.2.1.85 Dimethyl- maleate hydratase dmdB, dmdA (Eubacterium barkeri)

EC 4.2.1.89 L-carnitine dehydratase caiB (E. Coli) Swiss-Prot P31572

EC 4.2.1.105 2-hydroxyiso- flavanone dehydratase HIDH (Glycine max)

EC 4.2.1.111 1,5-anhydro-D- fructose dehydratase

EC 4.2.1.113 o-succinyl- benzoate synthase menC (Escherichia coli K12;Bacillus subtilis)

EC 4.2.1.2 Fumarate hydratase fumC (Escherichia coli K12), fumB(Escherichia coli K12), fumA (Escherichia coli K12), FH (Homo sapiens),fumC (Mycobacterium tuberculosis), (fumC): HP1325 (Helicobacter pylori26695; Methanococcus maripaludis, Desulfobacter hydrogenophilusUniProt:O25883, UniProt:O53446, UniProt:O66271, UniProt:O69294,UniProt:O84863, UniProt:O94552, UniProt:P05042, UniProt:P07343,UniProt:P08417, UniProt:P0AC33 UniProt:P10173, UniProt:P14407,UniProt:P14408, UniProt:P39461, UniProt:P93033 UniProt:Q7M4Z3,UniProt:Q9JTE3, UniProt:Q9JTR0, UniProt:Q04718, UniProt:Q43180,UniProt:Q51404, UniProt:Q55674, UniProt:Q58034, UniProt:Q58690,UniProt:Q60022

EC 4.2.1.- crotonobetainyl- CoA hydratase caiD (Escherichia coli K12)

EC 4.2.1.- 2-hydroxy- isoflavanone dehydratase HIDH (Glycine max)

EC 4.2.1.- 2-oxohept-3- enedioate- hydroxylation

e) Conversion of 6-Aminohex-2-Enoic Acid into Aminocaproic Acid

Pathways I, II, IV, VI, VII, and VIII conclude with the conversion of6-aminohex-2-enoic acid into aminocaproic acid. The reduction of(E)-6-aminohex-2-enoic acid to aminocaproic acid (a4→a5, enzymatic stepA5) is catalyzed by a polypeptide having a reductase or a dehydrogenaseactivity. An example of an enzyme catalyzing this reaction is anenoate-reductase capable of hydrogenating the α,β-carbon-carbon doublebond at the α,β-position next to a carboxylic acid group into acarbon-carbon single bond. Example of enzymes having a α,β enoatereductase activity can be found in US applications 20070117191 and20070254341. Other exemplary polypeptides having an enoate reductaseactivity are listed in Table 4. Preferably, the enoate reductase is a2-enoate reductase, a NADH-dependent fumarate reductase, a succinatedehydrogenase, a coumarate reductase, a β-nitroacrylate reductase, amethylacetate reductase, or a γ-butyrobetaine reductase.

TABLE 4 Desired substrate and product reaction EC number Name Gene name(organism) Protein accession number (organism)

EC 1.3.1.31 2-enoate reductase P11887 (Clostridium tyrobutyricum)

EC 1.3.1.6 NADH- dependent fumarate reductase

EC 1.3.99.1 Succinate dehydrogenase sdhA (Mycobacterium tuberculosis),(Methanococcus maripaludis) fumarate reductase (Aquifex pyrophilus)

EC 1.3.1.11 Coumarate reductase

EC 1.3.1.16 β-nitroacrylate reductase

EC 1.3.1.32 Maleyl acetate reductase pnpE (Pseudomonas sp. ENV2030);macA (Rhodococcus opacus); (Xanthobacter flavus); tftE (Burkholderiacepacia) Q45072 (Burkholderia cepacia)

EC 1.3.1.9 NADPH 2-enoyl CoA reductase enrA1 (Brassica napus)

EC 1.3.1.39 Enoyl-[acyl-carrier protein] reductase FASN (Homo sapiens)

EC 1.-.-.- N-ethylmaleimide reductase, FMN- linked Gene: nemA(Escherichia coli K12) Protein accession: P77258

EC 1.3.99.- Crotonobetaine reductase

f) Conversion of 6-amino-3-hydroxyhexanoic acid into6-amino-3-hydroxyhexanoyl-CoA

As described in pathways III and IV, 6-amino-3-hydroxyhexanoic acid isconverted to 6-amino-3-hydroxyhexanoyl-CoA. Although enzymes thatcatalyze the substrate to product conversion of6-amino-3-hydroxyhexanoic acid into 6-amino-3-hydroxyhexanoyl-CoA(enzymatic step A6) have not been described, one skilled in the art willappreciate that the 6-amino-3-hydroxyhexanoic acid substrate is similarto substrates listed in table 5 and is likely to be an acceptablesubstrate for enzymes having a CoA transferase or a CoA ligase activitylisted in Table 5. CoA-transferases catalyze reversible transferreactions of Coenzyme A groups from CoA-thioesters to free carboxylicacids. Most CoA transferases operate with succinyl-CoA or acetyl-CoA aspotential CoA donors and contain two different subunits.

TABLE 5 Desired substrate product reaction EC number Name Gene name(organism) Protein accession number

EC 6.2.1.3 fatty acyl-CoA synthetase fadD (Escherichia coli K12); fadK(Escherichia coli K12); ACSL1 (Homo sapiens); LACS7 (Arabidopsisthaliana col); LACS6 (Arabidopsis thaliana col); alkK (Pseudomonasoleovorans) UniProt:O15840, O30039, O51162 O51539, O60135, O81614,O83181, P18163, P30624, P33121, P33124, P39002, P39518, P44446, P47912,P69451, P69452 P73004, P94547 Q8JZR0, Q96338 Q96537, Q96538 Q9CHR0,Q9JTK0 Q9JYJ7, Q9RTR4 Q9RYK3, Q9T009 Q9T0A0, Q9X7Y5 Q9X7Z0, Q9YCF0Q9ZBW6, Q02602 Q10776

EC 6.2.1.3 acyl-CoA synthetase LACS2 (Arabidopsis thaliana col); LACS9(Arabidopsis thaliana col); LACS7 (Arabidopsis thaliana col); LACS6(Arabidopsis thaliana col)

EC 6.2.1.14 6 carboxy- hexanoate-CoA ligase bioW (Lysinibacillussphaericus); bioW (Bacillus subtilis)

EC 2.8.3.14 5 hydroxy- pentanoate CoA- transferase

EC 2.8.3.13 Succinate- hydroxymethyl- glutarate CoA- transferase

EC 2.8.3.- γ-butyrobetainyl- CoA: carnitine CoA transferase caiB(Escherichia coli K12)

g) Conversion of 6-amino-3-hydroxyhexanoyl-CoA into6-amino-hex-2-enoyl-CoA

In some embodiments, 6-amino-3-hydroxyhexanoyl-CoA is converted into6-amino-hex-2-enoyl-CoA (enzymatic steps A7, pathways III and IV).Although enzymes that catalyze this conversion have not been described,one will appreciate that 6-amino-3-hydroxyhexanoyl-CoA may likely be anacceptable substrate for the dehydratase or hydro-lyase enzymes listedin Table 3. In a preferred embodiment, the reaction is catalyzed byenoyl-CoA hydratase (EC 4.2.1.17), a methyl-glutaconyl-CoA hydratase (EC4.2.1.18), a crotonase (EC 4.2.1.55), a long-chain-enoylCoA hydratase(EC 4.2.1.74) or a 2-oxohept-3-enedioate hydrolase (EC 4.2.1-).

h) Conversion of 6-aminohex-2-enoyl-CoA to 6-aminohex-2-enoic acid

In Pathways IV and VIII, 6-aminohex-2-enoyl-CoA is converted to(E)-6-aminohex-2-enoic acid (b3 to b4 conversion, enzymatic step B4).Although enzymes that catalyze the substrate b3 to product b4 conversionof 6-aminohex-2-enoyl-CoA to (E)-6-aminohex-2-enoic acid have not beendescribed, one skilled in the art will appreciate that the6-aminohex-2-enoyl-CoA substrate is similar to glutaconyl CoA (seebelow) and the substrates listed in Table 5. Therefore6-aminohex-2-enoyl-CoA is likely to be an acceptable substrate forenzymes having a CoA-transferase activity such as those listed below orin Table 5. Coenzyme A-transferases are a family of enzymes with adiverse substrate specificity and subunit composition. The(E)-glutaconate:acetyl-CoA CoA-transferase (EC 2.8.3.12) which catalyzesthe following reaction may catalyze the substrate to product conversionof 6-aminohex-2-enoylCoA to (E)-6-aminohex-2-enoic acid:

The (E)-glutaconate:acetyl-CoA CoA-transferase enzyme is part ofglutamate degradation V (via hydroxyglutarate) pathway and consists oftwo different subunits (Subunit A and B) encoded by the GctA and GctBgenes (UniProt:Q59111 and UniProt:Q59112).i) Conversion of 6-aminohex-2-enoyl-CoA into 6-aminohexanoyl-CoA

In some embodiments, and more specifically in pathways III, V and IX,6-aminohex-2-enoyl-CoA is converted into 6-aminohexanoyl-CoA (enzymaticstep B5). Although this conversion has not been described, one willappreciate that the dehydrogenase enzymes listed in Table 4 may be ableto convert 6-aminohex-2-enoyl-CoA to 6-aminohexanoyl-CoA. In a preferredembodiment, the enzymatic step B5 is catalyzed by a butyryl-CoAdehydrogenase (EC 1.3.2.1, bcd, Megasphaera elsdenii, Clostridiumacetobutylicum), a Glutaryl-CoA dehydrogenase (EC 1.3.99.7),2-methylacyl-CoA dehydrogenase (EC 1.3.99.10, acdH Streptomycesavermitilis), 2-methyl branched-chain acyl-CoA dehydrogenase (acdH(Streptomyces avermitilis), Acadsb (Rattus norvegicus), or acrotonobetaine reductase (EC 1.3.99.-, caiA, Escherichia coli K12). Inanother preferred embodiment, the dehydrogenase of enzymatic step B5 isan acyl-CoA dehydrogenase. Acyl-CoA dehydrogenases are a class ofenzymes (EC 1.3.99.3, encoded by fadE in Escherichia coli K12,isovaleryl-CoA dehydrogenase encoded by aidB in Escherichia coli K12,encoded by acdB in Clostridium difficile) which catalyze the initialstep in each cycle of fatty acid β-oxidation in the mitochondria ofcells. Their action results in the introduction of a trans double-bondbetween C2 and C3 of the acyl-CoA thioester substrate. FAD is a requiredco-factor in the mechanism in order for the enzyme to bind to itsappropriate substrate.

j) Conversion of 1-amino-6-hexanoyl-CoA into 6-aminohexanoic acid

In enzymatic step (B6), 1-amino-6-hexanoyl-CoA is converted to6-aminohexanoic acid (Pathways III, V, and IX). Although enzymes thatcatalyze this substrate conversion have not been described, one skilledin the art will appreciate that the substrate 1-amino-6-hexanoyl-CoA issimilar to the substrates listed in Table 5. Therefore,1-amino-6-hexanoyl-CoA is likely to be an acceptable substrate forenzymes having a CoA-transferase activity, such as those listed belowand in Table 5. In a preferred embodiment, the CoA-transferase enzymesbelong to the EC 2.8.1.x class. A preferable CoA transferase is a2-hydroxyisocaproate CoA transferase. 2-hydroxyisocaproate CoAtransferase has been shown to catalyze the reversible conversion of2-hydroxyisocaproate into 2-hydroxyisocaproate CoA in the reduction ofL-Leucine to isocaproate pathway in Clostridium difficile (Kim et al.,Applied and Environmental Microbiology, 2006, Vol. 72, pp 6062-6069).

k) Conversion of (S)-3,6-diaminohexanoic acid to(S)-3,6-diaminohexanoyl-CoA

According to pathways I and V, (S)-3,6-diaminohexanoic acid is convertedto 3,6-diaminohexanoyl-CoA (enzymatic step B2). Although enzymes thatcatalyze the substrate to product conversion of (S)-3,6-diaminohexanoicacid to 3,6-diaminohexanoyl-CoA have not been described, one skilled inthe art will appreciate that the (S)-3,6-diaminohexanoic acid substrateis similar to substrates listed in Table 5. Therefore,(S)-3,6-diaminohexanoic acid is likely to be an acceptable substrate forenzymes having a CoA transferase or a CoA ligase activity, such as theenzymes listed in Table 5. CoA-transferases catalyze reversible transferreactions of Coenzyme A groups from CoA-thioesters to free carboxylicacids. Most CoA transferases operate with succinyl-CoA or acetyl-CoA aspotential CoA donors and contain two different subunits.

l) Conversion of 3,6-diaminohexanoyl-CoA into 6-aminohex-2-enoyl-CoA

In enzymatic step B3 (Pathways 1 and V), 3,6-diaminohexanoyl-CoA isconverted to 6-aminohex-2-enoyl-CoA. Although enzymes that catalyze thesubstrate to product conversion of 3,6-diaminohexanoyl-CoA to6-aminohex-2-enoyl-CoA have not been described, one skilled in the artwill appreciate that the 3,6-diaminohexanoyl-CoA substrate is similar tosubstrates listed in Table 6. Therefore, 3,6-diaminohexanoyl-CoA islikely to be an acceptable substrate for enzymes having a deaminaseactivity, such as those listed in Table 6.

TABLE 6 Desired substrate and product reaction EC number Name Gene name(organism) Protein accession number

EC 4.3.1.14 3-amino-butyryl-CoA ammonia-lyase

EC 4.3.1.6 beta-alanyl-CoA ammonia-lyase kal (Fusobacterium nucleatum)(Clostridium SB4)

m) Conversion of (S)-3,6-diaminohexanoic acid into 6-aminohex-2-enoicacid

In some embodiments, (S)-3,6-diaminohexanoic acid is directly convertedto 6-aminohex-2-enoic acid (enzymatic step B′, Pathway VI) by apolypeptide having a deaminase activity. Although enzymes that catalyzethe substrate to product conversion of 3,6-diaminohexanoate to6-aminohex-2-enoyl have not been described, one skilled in the art willappreciate that the 3,6-diaminohexanoate substrate is similar toL-aspartate or (2S,3S)-3-methylaspartate. Therefore,3,6-diaminohexanoate is likely to be an acceptable substrate forMethylaspartate ammonia-lyase isolated for example from Citrobacteramalonaticus, Citrobacter freundii, Clostridium tetanomorphum, orMorganella morganiithat which catalyzes the following reaction:

n) Conversion of lysine into 6-amino-2-oxohexanoic acid

Aspects of the invention provide metabolic pathways for the productionof aminocaproic acid from lysine via a 6-amino-2-hydroxyhexanoic acidintermediate (Pathways VII, VIII and IX). As depicted in FIG. 2, lysinecan be first converted to 6-amino-2-oxohexanoic acid (enzymatic step C1)and the resulting 6-amino-2-oxohexanoic acid is then converted to6-amino-2-hydroxyhexanoic acid (enzymatic step C2). In some embodiments,the resulting 6-amino-2-hydroxyhexanoic acid can be then converted to(E)-6-aminohex-2-enoic acid (enzymatic step C3) and finally theresulting ((E)-6-aminohex-2-enoic acid is converted to aminocaproic acid(as described in enzymatic step A5). In other embodiments,6-amino-2-hydroxyhexanoic acid is first converted to6-amino-2-hydroxyhexanoyl-CoA (enzymatic step C4), then into6-aminohex-2-enoyl-CoA (enzymatic step C5) and subsequently to6-aminohexanoic acid as described above (enzymatic steps B5 followed byB6 or enzymatic steps B4 followed by A5).

In some embodiments (enzymatic step C1), lysine is first converted to6-amino-2-oxohexanoic acid (c1) by an aminotransferase enzyme (EC2.6.1.x) or a dehydrogenase (EC 1.4.1.x or EC 1.4.3.x). In someembodiments, the pathway involves a lysine racemase that convertsL-lysine to D-lysine, which is then converted to cyclicΔ¹-piperideine-2-carboxylate intermediate which is then spontaneouslyconverted to 6-amino-2-oxohexanoic acid in the presence of water (seeFIG. 4). Lysine racemase enzymes include enzymes of the EC 5.1.1.5class. In other embodiments, L-lysine is directly converted to cyclicΔ¹-piperideine-2-carboxylate by a lysine dehydrogenase (EC 1.4.1.15,isolated from, for example, Agrobacterium tumefaciens or Candidaalbicans). In another embodiment, L-lysine is converted to6-amino-2-oxohexanoic acid by a lysine-α-oxidase (EC 1.4.3.14,Trichoderma viride 14). Yet in other embodiments, enzymes listed inTable 7 may be used or engineered to catalyze the conversion of L-lysineto 6-amino-2-oxohexanoic acid.

TABLE 7 Desired substrate- product reaction EC number Name Gene name(organism) Protein accession number

EC 2.6.1.35 glycine-oxaloacetate transaminase Micrococcus denitrificans,Rhodobacter capsulatus strain E1F1, Rhodo- pseudomonas acidophila

EC 2.6.1.42 Branched-chain amino acid aminotransferase (transaminase B)ilvE (Escherichia coli K12); ilvE (Methano- thermobacterthermautotrophics); ilvE (Methanococcus aeolicus); BAT1 (Saccharomycescerevisiae S288C); ilvE (Pseudomonas aeruginosa) UniProt: O14370,UniProt: O32954, UniProt: O86505, UniProt: P0AB80, UniProt: P38891,UniProt: P47176, UniProt: Q9PIM6

EC 2.6.1.21 D-alanine aminotransferase Bacillus sp., Bacillus subtilis,Pisum sativum Rhizobium japonicum

EC 2.6.1.8 Diamino-acid aminotransferase Aspergillus fumigatus,Escherichia coli, Mus musculus

EC 2.6.1.19 β-alanine aminotransferase Rattus norvegicus

EC 2.6.1.67 2-aminohexanoate aminotransferase Candida guilliermondiivar. membranaefaciens

EC 2.6.1.22 L-3- aminoisobutyrate aminotransferase Rattus norvegicus;Sus scrofa

EC 2.6.1.12 Alanine-oxo-acid aminotransferase Pseudomonas sp.; Brucellaabortus

EC 2.6.1.40 β-amino- isobutyrate-- pyruvate transaminase Caviaporcellus, Homo sapiens, Rattus norvegicus, Sus scrofa

EC 1.4.1.15 Lysine dehydrogenase Piperidine-2- carboxylate isspontaneously converted to 6- amino-2- oxohexanoic acid.

EC 1.4.1.2 Glutamate dehydrogenase GDH2, GDH1 (Arabidopsis thalianacol), gdhA (Peptoniphilus asaccharolyticus, Halobacterium salinarum),gdh (Thermotoga maritima), GLUD1, GLUD2 (Homo sapiens), Clostridiumpropionicum, Bacillus subtilis

EC 1.4.1.16 meso- diaminopimelate D-dehydrogenase ddh (Corynebacteriumglutamicum), dapdh (Lysinibacillus sphaericus)

o) Conversion of 6-amino-2-oxohexanoic acid is Converted to6-amino-2-hydroxyhexanoic acid

According to enzymatic step C2,6-amino-2-oxohexanoic acid (c1) isconverted to 6-amino-2-hydroxyhexanoic acid (c2, Pathways VII, VIII andIX). In some embodiments, this reaction is catalyzed by a reductase ordehydrogenase enzyme. Although there are no known enzymes that catalyzethis specific reaction, dehydrogenase enzymes capable of reducing thecarboxylic group on c2 to a secondary alcohol may be considered.Examples of enzymes having, or which can be engineered to have, a6-amino-2-oxohexanoic acid dehydrogenase activity can be isolated from avariety of sources and include, but are not limited to, the enzymeslisted in Table 8.

TABLE 8 Desired substrate- product reaction EC number Name Gene name(organism) Protein accession number

EC 1.1.1.5 diacetyl reductase budC (Enterobacter aerogenes); budC(Klebsiella pneumoniae); butA (Corynebacterium glutamicum)

EC 1.1.1.26 Glycolate reductase

EC 1.1.1.27 L-lactate dehydrogenase ldh (Streptococcus mutans); ldh2(Bifidobacterium longum); Ldha (Rattus norvegicus), ldh (Lactobacilluscasei); ldh (Mycoplasma pneumoniae M129); ldh (Thermotoga maritima)UniProt: O23569, O81272, P00336, P00337, P00338, P00339, P00340, P00341,P00342, P00343, P00344, P00345, P04034, P04642, P06150, P06151, P07195,P07864, P0C0J3, P10655, P13490, P13491, P13714, P13715, P13743, P14561,P16115, P16125, P19629, P19858, P19869, P20373, P20619, P22988, P22989,P26283, P29038, P33232, P33571, P42119, P42120, P42121, P42123, P46454,P47698, P50933, P56511, P56512, Q7M1E1, Q96570, Q9CGG8, Q9CII4, Q9PNC8,Q01462, Q06176, Q07251, Q27888, Q48662, Q59244, Q60009, Q60176, Q62545,Q96569, Q9SBE4, Q9ZRJ5

EC 1.1.1.37 malate dehydrogenase mdh (Escherichia coli K12); MDH1 (Homosapiens), MDH2 (Homo sapiens), malate dehydrogenase (Propionibacteriumfreudenreichii subsp. shermanii); malate dehydrogenase 1 MDH1 (Homosapiens); MDH1 (Sus scrofa); mdh (Mycobacterium tuberculosis); malatedehydrogenase (Methanococcus maripaludis), malate dehydrogenase (Aquifexpyrophilus); mdh (NAD-linked) (Methylobacterium extorquens AM1) UniProt:O24047, O48903, O48904, O48905, O48906, O65363, O65364, O81278, O81279,O81609, P04636, P10887, P11386, P11708, P14152, P16142, P17505, P17783,P19446, P19977, P19978, P19979, P19980, P19981, P19982, P19983, P22133,P25077, P32419, P33163, P44427, P46487, P46488, P49814, P50917, P58408,P93106, Q7M4Y9, Q7M4Z0, Q8R1P0, Q93ZA7, Q9PHY2, Q9SN86, Q9XTB4, Q9ZP05,Q9ZP06, Q04820, Q07841, Q42686, Q42972, Q43743, Q43744, Q49981, Q55383,Q58820, Q59202

EC 1.1.1.93 tartrate dehydrogenase Pseudomonas putida

EC 1.1.1.110 Indole-3-lactate dehydrogenase Clostridium sporogenes;Lactobacillus casei

EC 1.1.1.111 3-(imidazol-5- yl)lactate dehydrogenase imidazole-lactateoxidase (Comamonas testosteroni); imidazol- pyruvate reductase(Escherichia coli B)

EC 1.1.1.125 2-deoxy-D- gluconate 3-dehydrogenase kduD (Escherichiacoli) UniProt P37769 (Escherichia coli)

EC 1.1.1.167 Hydroxymalonate dehydrogenase

EC 1.1.1.169 2-dehydro- pantoate 2- reductase panE (Escherichia coliK12) UniProt: Q9CFY8 (Streptococcus lactis), Q9V0N0 (Pyrococcus abyssi)

EC 1.1.1.172 2-oxoadipate reductase

EC 1.1.1.215 Gluconate 2- dehydrogenase ghrB (Escherichia coli K12)

EC 1.1.1.222 (R)-4- (hydroxy- phenyl)lactate dehydrogenase

EC 1.1.1.237 hydroxy- phenylpyruvate reductase HPPR (Solenostemonscutellarioides)

EC 1.1.1.272 (R)-2-hydroxyacid dehydrogenase

EC 1.1.1.285 3″-deamino-3″- oxonicotianamine reductase or deoxymugineicacid synthase ZmDMAS1 (Zea mays), TaDMAS1 (Triticum aestivum) hvDMAS1(Hordeum vulgare), OsDMAS1 (Oryza), 3″-deamino- 3″-oxonicoti- anaminereductase (Hordeum vulgare)

EC 1.1.1.- oxalosuccinate reductase icd (Hydrogenobacter thermophilus)

EC 1.1.99.2 α-ketoglutarate reductase serA (Escherichia coli K12)

EC 1.1.99.3 D-gluconate dehydrogenase (Pseudomonas fluorescens) UniProt:O34213, O34214, O34215, Q9PI91

EC 1.1.99.6 D-2-hydroxy-acid dehydrogenase Ddh (Zymomonas mobilis), ddh(Haemophilus influenzae); Synechocystis sp., GRHPR (Homo sapiens)UniProt: P30799, P44501, P74586, Q9UBQ7

p) Conversion of 6-amino-2-hydroxyhexanoic acid to(E)-6-amino-hex-2-enoic acid

In enzymatic step C3 (Pathway VIII), 6-amino-2-hydroxyhexanoic acid isconverted to (E)-6-amino-hex-2-enoic acid by an enzyme having adehydratase activity. Enzymatic reactions that produce enzymes having adehydratase activity are part of the lyase class that catalyzes theremoval of H₂O, leaving a double bond as depicted in FIG. 2.Polypeptides having dehydratase activity as well as nucleic acidencoding such polypeptides can be obtained from various speciesincluding, without limitation, Escherichia coli K12; Bacillus subtilis,Eubacterium barkeri, Glycine max, Anthracobia melaloma, Plicariaanthracina and Plicaria leiocarpa. Polypeptides having a dehydrataseactivity as well as nucleic acid encoding such polypeptides include, butare not limited to, enzymes listed in Table 3, and to enzymes EC4.2.1.x, for example, EC 4.2.1.67 (D-fuconate hydratase), EC 4.2.1.68(L-fuconate hydratase), EC 4.2.1.85 (dimethylmaleate hydratase), EC4.2.1.-(crotonobetainyl-CoA hydratase) and EC 4.2.1.2 (Fumaratehydratase).

q) Conversion of 6-amino-2-hydroxyhexanoic acid is first converted to6-amino-2-hydroxyhexanoyl-CoA

In some embodiments, 6-amino-2-hydroxyhexanoic acid is first convertedto 6-amino-2-hydroxyhexanoyl-CoA by a CoA transferase or a CoA ligase(enzymatic step C4, Pathways VIII and IX). Enzymes capable of catalyzingthis reaction can be any suitable enzyme having a CoA transferaseactivity or a CoA ligase activity and include, but are not limited to,the enzymes listed in Table 5.

r) Conversion of 6-amino-2-hydroxyhexanoyl-CoA to(E)-6-aminohex-2-enoyl-CoA

Subsequently, 6-amino-2-hydroxyhexanoyl-CoA can be converted to(E)-6-aminohex-2-enoyl-CoA by an enzyme having a dehydratase activity(enzymatic step C5, Pathways VIII and IX). Polypeptides having adehydratase activity as well as nucleic acids encoding such polypeptidesinclude, but are not limited to, the enzymes listed in Table 3 and thefollowing enzymes: EC 4.2.1.17 (enoyl-CoA hydratase or(3S)-3-hydroxyacyl-CoA hydro-lyase), EC 4.2.1.18 (methylglutaconyl-CoAhydratase or (S)-3-hydroxy-3-methylglutaryl-CoA hydro-lyase), EC4.2.1.55 (Crotonase or 3-hydroxybutyryl-CoA dehydratase), EC 4.2.1.54(lactyl-CoA dehydratase isolated in Clostridium propionicum), EC4.2.1.56 (itaconyl-CoA hydratase isolated in Pseudomonas sp.), and to EC4.2.1.-(2-Hydroxyglutaryl-CoA dehydratase). For example, the2-Hydroxyglutaryl-CoA dehydratase (HgdAB, CompD) catalyzes thesyn-elimination of water from (R)-2-hydroxyglutaryl-CoA to(E)-glutaconyl-CoA as shown below:

C. Engineered Pathways for the Production of Aminocaproic Acid from aNitrogen-Containing Heterocyclic Ring

Aspects of the invention relate to the bioproduction of C6 and C5difunctional alkanes from nitrogen containing heterocyclic rings. Anitrogen-containing heterocyclic ring includes but is not limited topiperidine, pyridine, picolinate and pipecolate. A preferablenitrogen-containing heterocyclic ring is L-2,3-dihydropicolinate (alsoknown as (S)-2,3-dihydropyridine-2,6-dicarboxylic acid),L-2,3,4,5,-terahydrodipicolinate (also know asΔ¹-piperideine-2,6-dicarboxylate or(S)-2,3,4,5-terahydropyridin-2,6-dicarboxylic acid),Δ¹-piperideine-2-dicarboxylate, Δ¹-piperideine-6-dicarboxylate,5,6-dihydropyridine-2-carboxylic acid, L-pipecolate and D-pipecolate. Onskilled in art will understand that some of these nitrogen-containingheterocyclic rings are intermediates metabolites in the L-lysinebiosynthesis pathway or in the L-lysine or D-lysine degradationpathways. For example, L-2,3-dihydropicolinate andΔ¹-piperideine-2,6-dicarboxylate are intermediate metabolites in thelysine biosynthesis pathways I, II, III, and VI. L-pipecolate,Δ¹-piperideine-6-carboxylate, and Δ¹-piperideine-2-carboxylate areintermediate metabolites in the L-lysine or D-lysine degradationpathway. In some embodiments, the bioconversion of lysine toΔ¹-piperideine-2-carboxylate is catalyzed by a lysine dehydrogenase(FIG. 4, EC 1.4.1.15).

Some aspects of the invention provide engineered metabolic pathways forthe production of 6-amino-2-oxohexanoic acid fromL-2-3-dihydrodipicolinate, an intermediate metabolite of the lysinebiosynthetic pathway. Six lysine biosynthetic pathways have beendescribed in bacteria, most algae, fungi and higher plants. Thesepathways are divided into the diaminopimelate pathways, and theα-aminoadipate pathways. In the pathways that belong to thediaminopimelate group, lysine is produced from aspartate (along withmethionine, threonine and isoleucine). All of these pathways share thefirst enzymatic steps, and in particular the three steps required forconversion of L-aspartate to L-2,3-dihydrodipicolinate (and totetrahydrodipicolinate). In the diaminopimelate pathway, L-aspartate isfirst converted to L-aspartyl-4-phosphate by an aspartate kinase (oraspartokinase), and then to L-aspartate semialdehyde (aspartatesemialdehyde dehydrogenase) and finally to L-2,3-dihydrodipicolinate(dihydrodipicolinate synthase). Polypeptides having an aspartate kinaseactivity may be isolated from a variety of sources. Some example ofsuitable aspartate kinases and genes encoding for aspartate kinases areavailable from a variety of sources including, but not limited to, lysC(Escherichia coli K12), thrA (Escherichia coli K12), metL (Escherichiacoli K12), AT3G02020 (Arabidopsis thaliana col), yclM (Bacillussubtilis), lysCβ, lysCα (Bacillus subtilis), lysCβ, lysCα(Corynebacterium glutamicum) and aspartokinase (Chromohalobactersalexigens). Polypeptides having an aspartate semialdehyde dehydrogenaseactivity, and genes encoding enzymes having an aspartate semialdehydedehydrogenase activity, may be isolated from a variety of sources suchas asd from Escherichia coli K12. Polypeptides having adihydropicolinate synthase activity, and genes encoding enzymes having adihydropicolinate synthase activity, may be isolated from a variety ofsources. Some example of suitable dihydrodipicolinate synthase, andgenes encoding for dihydrodipicolinate, are available from a variety ofsources and include, but are not limited to, dapA (Escherichia coliK12), dapA (Glycine max), dapA (Bacillus licheniformis) and dapA(Corynebacterium glutamicum).

One should appreciate that, according to some aspects of the invention,at least one of the genes for the lysine biosynthetic pathway or thelysine degradation pathway is functionally inactivated in the host cell.In a preferred embodiment, host cells may be microorganisms synthesizingless than 50%, less 40%, less than 30%, less than 20%, and preferablyless than 10% of the amount of lysine naturally formed under the sameconditions (e.g., in a host cell having an unmodified lysinebiosynthetic and/or degradation pathway). In some embodiments, C6 and C5difunctional alkanes are produced from L-2,3-dihydropicolinate and5,6-dihydropyridine-2-carboxylic acid. Preferably, at least one of thefollowing enzymes is inactivated at the genetic level:Dihydrodipicolinate reductase (EC 1.3.1.26, dapB gene),2,3,4,5-tetrahydropyridine-2-carboxylate N-succinyltransferase (alsoknown as Tetrahydrodipicolinate succinylase, EC 2.3.1.117, dapD gene),L,L-diaminopimelate aminotransferase (E.C. 2.6.1.83, AT4G33680 gene,Hudson et al, 2006, Plant Physiol. 140(1):292-301; the enzyme was shownto be involved in lysine biosynthesis under physiological conditions byfunctional complementation of the E. coli lysine biosynthesis doublemutant of dapD and dapE), or Tetrahydrodipicolinate N-acetyltransferase(EC 2.3.1.89, ykuQ (Bacillus subtilis)). In other embodiments, C6 and C5difunctional alkanes are produced from Δ¹-piperideine-2,6-dicarboxylateand preferably at least one of the following enzymes is inactivated atthe genetic level: 2,3,4,5-tetrahydropyridine-2-carboxylateN-succinyltransferase, L,L-diaminopimelate aminotransferase,Tetrahydrodipicolinate N-acetyltransferase, or Diaminopimelatedehydrogenase (EC 1.4.1.16, ddh (Corynebacterium glutamicum), dapdh(Lysinibacillus sphaericus)). Accordingly, aspects of the inventionprovide a recombinant microorganism having an engineered metabolicpathway for the production of C6 and C5 difunctional alkanes from L-2,3dihydropicolinate.

In some aspects of the invention, C5 and C6 difunctional alkanes areproduced from Δ¹-piperideine-2,6-dicarboxylate. In a preferredembodiment, 6-aminocaproate, adipic acid, 1,6-hexanediol and6-hydroxyhexanoate are produced (FIGS. 3A and 3B).

Some aspects of the invention provide engineered metabolic pathways(Pathway D, Pathway D′ and Pathway E, FIG. 3A) for the production of6-amino-2-oxohexanoic acid from L-2,3-dihydrodipicolinate.

-   -   a) As depicted in Pathway D (FIG. 3A) L-2,3,dihydrodipicolinate        is first converted to Δ¹-piperidine-2,6-dicarboxylate (also        known as L-2,3,4,5-tetrahydrodipicolinate, enzymatic step D1),        the resulting Δ¹-piperidine-2,6-dicarboxylate is converted to        L-α-amino-ε-ketopimelate (step D2), and the resulting        L-α-amino-ε-ketopimelate is converted to 6-amino-2-oxohexanoic        acid (enzymatic step D3). One should appreciate that        6-amino-2-oxohexanoic acid can be produced directly from lysine        by a L-lysine α-oxidase (FIG. 4, EC 1.4.3.14, Trichoderma viride        i4).    -   b) As depicted in Pathway D′ (FIG. 3A):        L-2,3-dihydrodipicolinate is first converted to        Δ¹-piperidine-2,6-dicarboxylate (also known as        L-2,3,4,5-tetrahydrodipicolinate, enzymatic step D1), the        resulting Δ¹-piperidine-2,6-dicarboxylate is then converted to        Δ¹-piperidine-2-carboxylate (enzymatic step D′2), and the        resulting Δ¹-piperidine-2-carboxylate is converted to        6-amino-2-oxohexanoic acid (step D′3). In an alternative        pathway, Δ¹-piperidine-2-carboxylate may be first converted to        L-pipecolate (enzymatic step D′4) and then to        6-amino-2-oxohexanoic acid (enzymatic step D′5).    -   c) As depicted in Pathway E (FIG. 3A): L-2,3-dihydrodipicolinate        is first converted to 5,6-dihydropyridine-2-carboxylic acid        (enzymatic step E1); the resulting        5,6-dihydropyridine-2-carboxylic acid is then converted to        (E)-6-amino-2-oxohex-3-enoic acid (step E2) and the resulting        (E)-6-amino-2-oxohex-3-enoic acid is converted to        6-amino-2-oxohexanoic acid (enzymatic step E3).

One will appreciate that the final steps of all pathways D, D′ and E maybe identical to the conversion of 6-amino-2-oxohexanoic acid toaminocaproic acid as described above (enzymatic step C2→C3→A5 orC2→C4→C5→B5→B6, or C2→C4→C5→B4→A5).

a) Conversion of L-2,3-dihydrodipicolinate toΔ¹-piperidine-2,6-dicarboxylate (enzymatic step D1)

In some embodiments, L-2,3-dihydrodipicolinate may be converted toΔ1-piperidine-2,6-dicarboxylate (or tetrahydropicolinate) by action ofan enzyme such as a dihydrodipicolinate reductase (belonging to EC1.3.1.26, enzymatic step D1) that catalyzes the following chemicalreaction in the lysine biosynthesis pathway:

Polypeptides having a dihydrodipicolinate reductase activity, and genesencoding enzymes having a dihydrodipicolinate reductase activity, may beisolated from a variety of sources and include, but are not limited to,dapB from Escherichia coli K12, dapB from Bacillus subtilis, dapB fromCorynebacterium glutamicum, dapB from Mycobacterium tuberculosis anddihydrodipicolinate reductase from Zea mays.

b) Conversion of Δ¹-piperidine-2,6-dicarboxylate to6-amino-2-oxohexanoic acid via a Δ¹-piperidine-2-carboxylateintermediate (enzymatic steps D′2→D′3)

In some embodiments, Δ¹-piperidine-2,6-dicarboxylate (ortetrahydropicolinate) is then converted to Δ¹-piperidine-2-carboxylate(or 3,4,5,6-tetrahydropyridine-2-carboxylic acid) by action of adecarboxylase. Although there are no known enzymes having aΔ¹-piperidine-2,6-dicarboxylate decarboxylase activity, polypeptidescapable of removing carboxyl groups from aromatic rings may be used. TheΔ¹-piperidine-2-carboxylate ring can then spontaneously open in thepresence of water to produce 6-amino-2-oxohexanoic acid as depictedbelow in the lysine degradation pathways V and VII.

Examples of enzymes having a decarboxylase activity include, but are notlimited to, 3-hydroxy-2-methylpyridine-4,5-dicarboxylate 4-decarboxylase(EC 4.1.1.51), benzoylformate decarboxylase (EC 4.1.1.7) and2,3-dihydrobenzoic acid decarboxylase (See U.S. Pat. No. 6,440,704).3-hydroxy-2-methylpyridine-4,5-dicarboxylate 4-decarboxylase has beenidentified in Mesorhizobium loti and in Pseudomonas sp. MA-1.

Benzoylformate decarboxylase (catalyzing the reaction as shown below)and the genes encoding such polypeptides may be found in a variety ofspecies including, but not limited to, Pseudomonas fluorescens andPseudomonas putida (gene mdlC, UniProt:P20906).

c) Conversion of Δ¹-piperidine-2,6-dicarboxylate to6-amino-2-oxohexanoic acid (enzymatic steps D2→D3)

In other embodiments, Δ¹-piperidine-2,6-dicarboxylate undergoesspontaneous dehydration to produce L-α-amino-ε-ketopimelate as in thelysine biosynthesis pathway III.

In one embodiment, L-α-amino-ε-ketopimelate is converted to6-amino-2-hexanoic acid by action of a decarboxylase, for example,diaminopimelate decarboxylase (EC 4.1.1.20), an enzyme shown to catalyzethe last reaction step in the lysine biosynthesis pathway:

Although enzymes that catalyze the substrate to product conversion of(S)-2-amino-6-oxoheptanedioic acid to 6-amino-2-oxohexanoic have notbeen described, one skilled in the art will appreciate that thesubstrate (S)-2-amino-6-oxoheptanedioic acid is similar to thesubstrates listed in Table 9. Therefore, (S)-2-amino-6-oxoheptanedioicacid is likely to be an acceptable substrate for enzymes having adecarboxylase activity, such as the enzymes listed in Table 9.

TABLE 9 Desired substrate- product reaction EC number Name Gene name(organism) Protein accession number

EC 4.1.1.20 diaminopimelate decarboxylase lysA (Escherichia coli K12,Bacillus subtilis), Arabidopsis thaliana col UniProt: O05321, UniProt:O27390, UniProt: O29458, UniProt: O67262, UniProt: P00861, UniProt:P09890, UniProt: P0A5M4, UniProt: P19572, UniProt: P23630, UniProt:P41023, UniProt: P44316, UniProt: P56129, UniProt: Q9CG26, UniProt:Q9JWA6, UniProt: Q9PII5, UniProt: Q55484, UniProt: Q58497.

EC 4.1.1.11 Aspartate 1- decarboxylase panD: Escherichia coli K12;Aquifex aeolicus; Helicobacter pylori; Mycobacterium tuberculosis;Neisseria meningitidis serogroup A; Neisseria meningitidis serogroup B;Campylobacter jejuni; Deinococcus radiodurans; Thermotoga maritime;Helicobacter pylori J99; Synechocystis sp. UniProt: O66773, UniProt:P0A790, UniProt: P52999, UniProt: P56065, UniProt: P65660, UniProt:Q9JU49, UniProt: Q9JZ56, UniProt: Q9PIK3, UniProt: Q9RWF1, UniProt:Q9X037, UniProt: Q9ZN30, UniProt: Q55382

EC 4.1.1.17 Ornithine decarboxylase ornithine decarboxylase,biosynthetic: speC (Escherichia coli K12); ornithine decarboxylase,degradative: speF (Escherichia coli K12); ODC1 (Homo sapiens); speC(Pseudomonas aeruginosa) UniProt: O15696, UniProt: O66940, UniProt:O69865, UniProt: P00860, UniProt: P08432, UniProt: P09057, UniProt:P11926, UniProt: P14019, UniProt: P24169, UniProt: P27116, UniProt:P27118, UniProt: P27119, UniProt: P27120, UniProt: P27121, UniProt:P41931, UniProt: P43099, UniProt: P44317, UniProt: P49725, UniProt:P50134, UniProt: P93351, UniProt: P93357, UniProt: Q9TZZ6, UniProt:Q9UQW9, UniProt: Q9X2I6, UniProt: Q84527

EC 4.1.1.15 Glutamate decarboxylase gadA (Escherichia coli K12); gadB(Escherichia coli K12); GAD1 (Saccharomyces cerevisiae S288C); GAD2(Homo sapiens); GAD1 (Homo sapiens); GAD2 (Arabidopsis thaliana col);GAD1 (Arabidopsis thaliana col) UniProt: O81101, UniProt: P14748,UniProt: P18088, UniProt: P20228, UniProt: P48318, UniProt: P48319,UniProt: P48320, UniProt: P48321, UniProt: P69908, UniProt: P69910,UniProt: Q9CG20, UniProt: Q05329, UniProt: Q05683, UniProt: Q07346,UniProt: Q24062, UniProt: Q49854, UniProt: Q49855, UniProt: Q49863,UniProt: Q59956, UniProt: Q99259

EC 4.1.1.19 Arginine decarboxylase arginine decarboxylase, biosynthetic:speA (Escherichia coli K12); arginine decarboxylase, degradative: adiA(Escherichia coli K12); speA (Pseudomonas aeruginosa; Bacillus subtilis)UniProt: O04429, UniProt: O23141, UniProt: O24128, UniProt: O64453,UniProt: O81161, UniProt: O81177, UniProt: P21170, UniProt: P22220,UniProt: P28629, UniProt: P49726, UniProt: P72587, UniProt: P74576,UniProt: Q96412, UniProt: Q9JT25, UniProt: Q9PPF5, UniProt: Q9SI64,UniProt: Q39827, UniProt: Q43075

EC 4.1.1.14 Valine decarboxylase panD

d) Conversion of L-2,3-dihydropicolinate to5,6-dihydropyridine-2-carboxylate

In an alternative pathway, 6-amino-2-oxohexanoic acid is produced bydecarboxylation of L-2,3-dihydropicolinate to5,6-dihydropyridine-2-carboxylate (enzymatic step E1), followed by aspontaneous hydration of 5,6-dihydropyridine-2-carboxylate to(E)-6-amino-2-oxohex-3-enoic acid (Step E2), and a subsequentdehydrogenation of (E)-6-amino-2-oxohex-3-enoic acid to6-amino-2-oxohexanoic acid (enzymatic step E3) as depicted in FIG. 3A.

In some embodiments, L-2,3-dihydropicolinate is first converted to5,6-dihydropyridine-2-carboxylate as depicted below:

There are no enzymes known in the art that catalyze the substrateL-2,3-dihydropicolinate to produce 5,6-dihydropyridine-2-carboxylate.However, there are a few decarboxylases that are able to decarboxylatering mounted carboxylic acids. One skilled in the art would appreciatethat it is likely that these enzymes may have, or could be engineered tohave, activity towards L-2,3-dihydropicolinate. Examples of suchdecarboxylases include, but are not limited to, benzoylformatedecarboxylase (EC 4.1.1.7), 3-hydroxy-2-methylpyridine-4,5-dicarboxylatedecarboxylase (EC 4.1.1.51), pyrrole-2-carboxylase (EC 4.1.1.-), gallatedecarboxylase (4.1.1.59), Dopa decarboxylase (EC 4.1.1.28),Phenylpyruvate decarboxylase (EC 4.1.1.43), 4,5-dihydroxyphthalatedecarboxylase (EC 4.1.1.55), 5-dihydroxybenzoate decarboxylase (EC4.1.1.62), Uracil-5-carboxylate decarboxylase (EC 4.1.1.66), andCis-1,2-dihydroxycyclohexa-3,5-diene-1-carboxylate dehydrogenase (EC1.3.1.25). In a subsequent step, the 5,6-dihydropyridine-2-carboxylicacid ring is opened in the presence of water to produce(E)-6-amino-2-oxohex-3-enoic acid. This reaction is similar to thehydration of 2,3,4,5-terahydropyridine-2-carboxylic acid (in the lysinedegradation pathway) and is likely to be spontaneous in the presence ofwater.

e) Conversion of (E)-6-amino-2-oxohex-3-enoic acid is converted to6-amino-2-oxohexanoic acid

In a subsequent step (E3), the (E)-6-amino-2-oxohex-3-enoic acid isconverted to 6-amino-2-oxohexanoic acid. Although there are no enzymesknown in the art that catalyze the substrate to product conversion, theenzymes listed in Table 4 may also have an activity towards(E)-6-amino-2-oxohex-3-enoic acid or could be engineered to catalyze thereaction. Theses enzymes include, but are not limited to, 2-enoatereductase (EC 1.3.1.31), NADH-dependent fumarate reductase (EC 1.3.1.6),coumarate reductase (EC 1.3.1.11), β-nitroacrylate reductase (EC1.3.1.16), Maleylacetate reductase (EC 1.3.1.32), N-ethylmaleimidereductase (EC 1.-.-.-) and EC 1.3.99.-. One should appreciate that(E)-6-amino-2-oxohex-3-enoic acid could first be converted to(E)-6-amino-2-oxohex-3-enoyl-CoA, then to (E)-6-amino-2-oxohexanoyl-CoA,and then to (E)-6-amino-2-hydroxyl-hexanoyl-CoA. The final steps may beidentical to the ones detailed in Pathways VIII and IX.

f) Conversion of lysine to 6-amino-2-oxohexanoic acid

One should appreciate that lysine may be directly converted to6-amino-2-oxohexanoic acid by action of L-lysine oxidase as described inthe lysine degradation pathway VII (EC 1.4.1.3.14) (FIG. 4). TheL-lysine oxidase has been described in strains of the ascomycetesTrichoderma viride and Trichoderma harzianum.

D. Engineered Pathway for the Bioproduction of adipic acid,6-hydroxyhexanoate and 1,6-hexanediol from Nitrogen-ContainingHeterocyclic Rings.

Aspects of the invention relate to engineered metabolic pathways for thebioproduction of adipic acid, 1,6-hexanediol and 6-hydroxyhexanoate fromnitrogen-containing heterocyclic rings. One aspect of the inventionprovides engineered metabolic pathways for the bioproduction of adipicacid, 6-hydroxyhexanoate (6HH) and 1,6-hexanediol fromnitrogen-containing heterocyclic rings. In some aspects of theinvention, engineered metabolic pathways for the production of2-oxoadipate semialdehyde as a metabolite intermediate for theproduction of adipic acid, 6-hydroxyhexanoate and/or 1,6-hexanediol areprovided. Accordingly, aspects of the invention provide recombinantmicroorganisms having an engineered pathway for the production of adipicacid. Other aspects of the invention provide a recombinant microorganismhaving an engineered pathway for the production of 6-hydroxyhexanoate.Yet other aspects of the invention provide a recombinant microorganismhaving an engineered pathway for the production of 1,6-hexanediol.

In a preferred embodiment, the C6 difunctional alkanes are produced fromL-pipecolate. L-pipecolate is an intermediate of the L-lysine andD-lysine degradation pathway. L-pipecolate may also be bioproducedaccording to Pathway D′ as describe herein. According to some aspects ofthe invention, L-pipecolate is converted toΔ¹-piperideine-6-L-carboxylate, and then Δ¹-piperideine-6-L-carboxylateis converted to L-2-aminoadipate-6-semialdehyde;L-2-aminoadipate-6-semialdehyde is optionally converted toL-2-aminoadipate. One skilled in the art would appreciate that this setof reactions is part of the lysine degradation pathway. In someembodiments, L-pipecolate is converted to Δ¹-piperideine-6-L-carboxylateby action of L-pipecolate dehydrogenase or L-pipecolate oxidase.L-pipecolate dehydrogenases and L-pipecolate oxidase are known in theart and are part of the lysine degradation pathway. Examples ofL-pipecolate oxidases include, but are not limited to, EC 1.5.99.3(Pseudomonas putida) and EC 1.5.3.7. (Schizosaccharomyces pombe,Arabidopsis thaliana, Homo sapiens). In a preferred embodiment, theenzyme is L-pipecolate dehydrogenase with EC number 1.5.99.3.

The reactions catalyzed by L-pipecolate oxidase or dehydrogenase areshown below:

Δ¹-piperideine-6-L-carboxylate reacts with water to form spontaneouslyL-2-aminoadipate-6-semialdehyde. Δ¹-piperideine-6-L-carboxylate andL-2-aminoadipate-6-semialdehyde can then be converted to 2-aminoadipateby an L-aminoadipate-semialdehyde dehydrogenase or an oxido-reductase(EC 1.2.1.31), the oxidizing equivalent being supplied in the form of anoxidized nicotinamide cofactor NAD or NADH as detailed below:

L-aminoadipate-semialdehyde dehydrogenase or oxido-reductase have beendescribed in a variety of species including Pseudomonas putida (encodedby amaA), Streptomyces clavuligerus (encoded by pcd), Flavobacteriumlutescens (encoded by pcd), Homo sapiens, Mus musculus, Rattusnorvegicus, Schizosaccharomyces pombe, Acremonium chrysogenum,Penicillium chrysogenum and Candida albicans.

One should appreciate that 2-aminoadipate can be converted to adipicacid by removal of the α-amino group as described for the deamination oflysine in Pathways I through IX. Alternatively, 2-aminoadipate may beconverted to α-ketoadipate by a α-aminoadipate aminotransferase (EC2.6.1.39, gene aadat, lysN) or a diaminopimelate dehydrogenase (EC1.4.116, gene ddb or Dapdh) and subsequently to adipic acid as describedin pathway VII through IX.

Yet in another embodiment, L-2-aminoadipate-6-semialdehyde is convertedto adipate semialdehyde by removal of the α-amino group as described inPathways I through IX. Adipate semialdehyde can then finally beconverted to 1,6-hexanediol or 6-hydroxyhexanoate by an aldehydedehydrogenase and/or an alcohol dehydrogenase.

Aldehyde dehydrogenases catalyze the conversion of the aldehydefunctional group into an alcohol functional group. Alcoholdehydrogenases (ADHs) (EC 1.1.1.1 and EC 1.1.1.2) catalyze thereversible reduction of ketones and aldehydes to alcohols with thereduction of NAD+ to NADH. In some embodiments, the alcoholdehydrogenase includes, but is not limited, to adhA or adhB (from Z.mobilis), butanol dehydrogenase (from Clostridium acetobutylicum),propanediol oxidoreductase (from E. coli), and ADHIV alcoholdehydrogenase (from Saccharomyces), ADH6 (from S. cerevisiae). In someembodiments, the hydro-carboxylic acid is subjected to dehydrogenationusing an alcohol dehydrogenase or an aldehyde dehydrogenase to produce1,6-hexane diol. Aldehyde NAD(+) dehydrogenase activity and alcoholNAD(+) dehydrogenase activities can be carried out by two differentpolypeptides, or carried out by a single polypeptide, such as amulti-functional aldehyde-alcohol dehydrogenase (EC 1.2.1.10) from E.coli (Goodlove et al. Gene 85:209-14, 1989; GenBank Accession No.M33504). Polypeptides having aldehyde dehydrogenase (NAD(P)+) (EC1.2.1.-) or aldehyde dehydrogenase (NAD(+)) (EC 1.2.1.3) activity can beused in combination with an alcohol dehydrogenase to reduce theremaining carboxylic acid to an alcohol, yielding a diol. Nucleic acidsencoding such polypeptides can be obtained from various speciesincluding, without limitation, S. cerevisiae.

E. Engineered Pathways for the Production C6 difunctional from L-lysine

1. Bioproduction of adipic acid, 6-hydroxyhexanoate and 1,6-hexanediol

Some aspects of the invention provide biosynthetic pathways for theproduction of adipic acid, 6-hydroxyhexanoate, 1,6-hexanediol fromlysine and from 2-oxoadipate semialdehyde. All these pathways share thefirst enzymatic step in which lysine is first converted to2-amino-adipate-6-semialdehyde (see FIG. 5).

In one embodiment, in enzymatic step 5a or 5m, lysine is first convertedto α-aminoadipate semialdehyde (also known as2-aminoadipate-6-semialdehyde), the resulting α-aminoadipatesemialdehyde is then converted to 2-oxoadipate semialdehyde (enzymaticstep 5b). In the initial step 5a, lysine is converted to α-aminoadipatesemialdehyde by action of an enzyme having a lysine amino-transferaseactivity. There are at least two known types of biochemical reactionsthat can catalyze the substrate to product conversion of lysine toα-aminoadipate semialdehyde. The first enzyme is a lysineamino-transferase (enzymatic step 5a, EC 2.6.1.36).

Enzymes catalyzing this substrate to product conversion are well knownand participate in the lysine degradation pathway VI and employpyridoxal phosphate as a cofactor. L-lysine 6-aminotransferase has beendemonstrated in yeasts and bacteria. Examples of suitableL-aminotransferase enzymes are available from a variety of sources, forexample, Nocardia lactamdurans (UniProt accession number Q05174),Flavobacterium lutescens (gene lat, Fujii et al., 2000, J. Biochem.,128(3);391-7), Candida utilis, Streptomyces clavuligerus (UniProt:Q01767, gene lat), Rhodococcus sp., Mycobacterium marinum, Mycobacteriumulcerans, Saccharopolyspora erythraea, Frankia alni and Frankia sp.

The second known enzyme capable of catalyzing the substrate to productconversion of lysine to α-aminoadipate semialdehyde isL-lysine-ε-dehydrogenase (enzymatic step 5m). L-lysine ε-dehydrogenasecatalyzes the oxidative deamination of the lysine epsilon amino group inan NAD+-dependent reaction (EC 1.4.18; Lysine degradation pathway VIII):

Examples of suitable L-lysine ε-dehydrogenase enzymes are available froma variety of sources, for example, Agrobacterium tumefaciens andGeobacillus stearothermophilus (gene: lysDH)

As illustrated in FIG. 5, 2-aminoadipate-6-semialdehyde can then beconverted to adipate semialdehyde (enzymatic step 5b followed byenzymatic step 5g or enzymatic step 5f) for the production of adipicacid, 6HH, or 1,6-hexanediol.

In a first embodiment, adipate semialdehyde is produced via anengineered pathway comprising enzymatic steps 5b and 5g. In theenzymatic step 5b (FIG. 5), 2-aminoadipate-6-semialdehyde is convertedto 2-oxoadipate semialdehyde by action of an enzyme having a2-aminoadipate activity. There are two known types of biochemicalreactions that could affect the substrate to product conversion, namelythe 2-aminoadipate aminotransferase (EC 2.6.1.39) and the Kynurenineaminotransferase II (EC 2.6.1.7) as depicted below.

Enzymes of the EC 2.6.1.39 class catalyze this substrate to productconversion and participate in the lysine degradation II, lysinedegradation V, lysine biosynthesis IV and lysine biosynthesis Vpathways. Examples of suitable α-aminoadipate aminotransferase enzymesare available from a variety of sources, for example, Thermusthermophilus (gene LysN, UniProt: Q5SL82, Q7×567, Q72LL6, Miyazaki etal., 2004, Microbiology 150; 2327-34), Thermus aquaticus (UniProtB4CM67), Rattus Norvegicus (Aadat, Kat2), and Homo sapiens (UniProtQ8N5Z0).

Kynurenine aminotransferase or Aspartate aminotransferase (Enzyme EC2.6.1.7, AspC, Escherichia coli K12) is a multifunctional enzyme thatcatalyzes the synthesis of aspartate, phenylalanine and other compoundsvia a transamination reaction.

In subsequent step 5g, 2-ketopadipate semialdehyde is converted toadipate semialdehyde by first action of a dehydrogenase (enzymatic step5g1) to convert the ketone group to a secondary alcohol group(2-hydroxyadipate semialdehyde), followed by action of a dehydratasethat catalyzes the conversion of the secondary alcohol group to analkene (enzymatic step 5g2), and finally followed by the action of adehydrogenase (enzymatic step 5g3) that catalyzes the conversion of thealkene to an alkane. In some embodiments, ketoadipate semialdehyde isfirst converted to the thiol ester by CoA transferase and then subjectedto enzymatic steps 5g1 to 5g3. Enzymes capable of catalyzing enzymaticsteps 5g include, but are not limited to, enzymes listed in enzymaticsteps C2, C4, C5, C3, B4, B5, B6 and A5.

Yet in other embodiments, 2-ketopadipate semialdehyde is first convertedto the corresponding acp-compound. In some embodiments, thedehydrogenase is a 3-oxoacyl-[acyl-carrier protein] reductase (EC1.1.1.100) shown to catalyze the following reaction in the fatty acidbiosynthesis superpathway:

In some embodiments, the dehydrogenase is aβ-ketoacyl-[acyl-carrier-protein] reductase (FabG gene from E. Coli),the fatty acid synthase (Fas2 gene from Saccharomyces cerevisiae) or thefatty acid synthase (FASN gene from Homo sapiens). In some embodiments,the dehydratase is a 3-hydroxyoctanoyl-[acyl-carrier-protein]dehydratase (EC 4.2.1.59) which catalyses the following reaction:

Examples of 3-hydroxyoctanoyl-[acyl-carrier-protein] dehydratasesinclude, but are not limited to, 3-hydroxyacyl-ACP dehydrase fromSpinacia oleracea, β-hydroxyacyl-ACP dehydrase (FabA gene from E. Coli),and β-hydroxyacyl-ACP dehydratase (FabZ gene from E. Coli).

In some embodiments, the second dehydrogenase is an enoyl acyl carrierprotein reductase (EC 1.3.1.9) such as enoyl-ACP reductase (fabI geneform E. Coli) shown to catalyze the following reaction:

In some embodiments, the first and the second dehydrogenase areidentical fatty acid synthases (Fas2 gene from Saccharomyces cerevisiaeor FASN gene from Homo sapiens).

In some embodiments, adipate semialdehyde can be used to produce1,6-hexanediol, 6HH, adipic acid, aminocaproic acid,hexamethylenediamine or 6-aminohexanol. In an exemplary embodiment,adipate semialdehyde is converted to 6HH by simple hydrogenation and thereaction is catalyzed by an alcohol dehydrogenase (EC 1.1.1.1). Thisenzyme belongs to the family of oxidoreductases, specifically thoseacting on the CH—OH group of donors with NAD⁺ or NADP⁺ as acceptors. Insome embodiments, a 6-hydroxyhexanoate dehydrogenase (EC 1.1.1.258) thatcatalyzes the following chemical reaction is used:6-hydroxyhexanoate+NAD⁺

6-oxohexanoate+NADH+H⁺

Other alcohol dehydrogenases include but are not limited to adhA or adhB(from Z. mobilis), butanol dehydrogenase (from Clostridiumacetobutylicum), propanediol oxidoreductase (from E. coli), and ADHIValcohol dehydrogenase (from Saccharomyces).

Yet in some embodiments, adipate semialdehyde is converted to6-hydroxyhexanoate by an alcohol dehydrogenase and then to1,6-hexanediol by action of an alcohol dehydrogenase or an aldehydedehydrogenase (enzymatic step 51). Alcohol dehydrogenases (ADHs) (EC1.1.1.1 and EC 1.1.1.2) catalyze the reversible reduction of ketones andaldehydes to alcohols with the reduction of NAD⁺ to NADH. In someembodiments, examples of alcohol dehydrogenases include but are notlimited to adhA or adhB (from Z. mobilis), butanol dehydrogenase (fromClostridium acetobutylicum), propanediol oxidoreductase (from E. coli),ADHIV alcohol dehydrogenase (from Saccharomyces), and ADH6 (from S.cerevisiae). Aldehyde NAD(+) dehydrogenase activity and alcohol NAD(+)dehydrogenase activities can be carried out by two differentpolypeptides, or carried out by a single polypeptide, such as amulti-functional aldehyde-alcohol dehydrogenase (EC 1.2.1.10) from E.coli (Goodlove et al. Gene 85:209-14, 1989; GenBank Accession No.M33504). Polypeptides having aldehyde dehydrogenase (NAD(P)+) (EC1.2.1.-) or aldehyde dehydrogenase (NAD(+)) (EC 1.2.1.3) activity, aswell as nucleic acids encoding such polypeptides, can be obtained fromvarious species including, without limitation, S. cerevisiae.

In a further alternative thereto, 2-aminoadipate-6-semialdehyde is firstconverted to 2-aminoadipate (enzymatic step 5c), the resulting2-aminoadipate is converted to 2-ketoadipate (enzymatic step 5d), andfinally to adipic acid (enzymatic step 5h).2-aminoadipate-6-semialdehyde is converted to 2-aminoadipate by actionof a L-aminoadipate-semialdehyde dehydrogenase (EC 1.2.1.31).

Examples of suitable L-aminoadipate-semialdehyde dehydrogenase enzymesare available form a number of sources, for example, Streptomycesclavuligerus (pcd), Flavobacterium lutescens (pcd), Acremoniumchrysogenum, Candida albicans, Candida maltosa, Homo sapiens,Kluyveromyces lactis, Mus musculus, Penicillium chrysogenum, Pichiaguilliermondii, Pseudomonas putida, Pseudomonas sp., Rattus norvegicus,Saccharomyces cerevisiae and Schizosaccharomyces pombe.

In enzymatic step 5d, the resulting 2-aminoadipate is converted to2-ketoadipate by action of a 2-aminoadipate aminotransferase (EC2.6.1.39) which participates in the amino-acid degradation pathway;L-lysine degradation via saccharopine pathway and in the glutaryl-CoAfrom L-lysine pathway. 2-ketoadipate is then converted to adipic acid(enzymatic steps 5h) by successive enzymatic steps described for theconversion of 2-ketoadipate semialdehyde to adipate semialdehyde.

In some embodiments, 2-amino adipate is converted to adipic acid bysuccessive enzymatic steps as described above for the conversion of1-aminoadipate-6-semialdehyde to adipate semialdehyde (enzymatic step5f, see Pathways I through IX).

2. Engineered Pathways for the Production of 6-Hydroxyhexanamine andHexamethylenediamine from Lysine

Aspects of the invention relates to the production of6-hydroxyhexanamine and/or hexamethylenediamine. Accordingly, aspects ofthe invention provide a recombinant microorganism having an engineeredpathway for the production of 6-hydroxyhexanamine andhexamethylenediamine. In a preferred embodiment, lysine is converted to2,6-diaminohexanal by action of an amino aldehyde dehydrogenase(enzymatic step 6a). 2,6-diaminohexanal can then be converted to6-aminohexanal by deamination (enzymatic step 6b) and subsequently to6-aminohexanol by an alcohol dehydrogenase (enzymatic step 6d). In someembodiments, 2,6-diaminohexanal is converted to 2-oxo-1,6-diaminohexane(enzymatic step 6c) by a semialdehyde aminomutase. Although enzymes thatcatalyze the 2,6-diaminohexanal to 2-oxo-1,6-diaminohexane conversionhave not been described, one should appreciate thatGlutamate-1-semialdehyde 2,1-aminomutase may catalyze this reaction.Glutamate-1-semialdehyde 2,1-aminomutase has been isolated in a varietyof species including, but not limited to, Escherichia coli,Synechococcus sp, Xanthomonas campestris, and Propionibacteriumfreudenreichii. Glutamate-1-semialdehyde 2,1-aminomutase genes include,but are not limited to, hemL (Escherichia coli K12, Salmonellatyphimurium) and GSA1 (Arabidopsis thaliana col). In a subsequent step,2-oxo-1,6-diaminohexane is then converted to 1,6-diaminohexane byremoval of the ketone group (enzymatic step 6h). In another embodiment,2-oxo-1,6-diaminohexane is first reduced to 2-hydroxy-1,6-diaminohexane(enzymatic step 6e) by an alcohol dehydrogenase, and then furtherreduced to 1,6-diamino-hexene (enzymatic step 60 which can then beconverted to hexamethylenediamine (enzymatic step 6e).

II. Engineered Pathways for the Production of C5 Difunctional Alkanes

Aspects of the invention relate to the bioproduction of C5 difunctionalalkanes. C5 difunctional alkanes of interest include5-hydroxypentanoate, glutarate, 1,5-pentanediol, 5-aminopentanoate,5-aminopentanol and cadaverine. Accordingly, aspects of the inventionprovide a recombinant microorganism having an engineered pathway for theproduction 5-hydroxypentanoate, glutarate, 1,5-pentanediol,5-aminopentanoate, 5-aminopentanol and cadaverine.

A. Engineered Pathways for the Production of C5 Difunctional Alkanesfrom Lysine.

Aspects of the invention relate to the bioproduction of C5 difunctionalalkanes from a C6 difunctional alkane. In a preferred embodiment, C5difunctional alkanes are produced from lysine as shown in FIG. 7.

Methods for producing cadaverine by introducing a lysine decarboxylationgene and/or a lysine-cadaverine antiporter gene into a lysine producingmicroorganism have been described (see for example JP 2002223770 andWO2008/092720). Lysine decarboxylase catalyzes the decarboxylation ofL-lysine into cadaverine. The enzyme has been classified as EC 4.1.1.18.The enzymes isolated from Escherichia coli having lysine decarboxylaseactivity are the cadA gene product and the ldc gene product.

As illustrated in FIG. 7, the C5 difunctional alkanes engineered pathwaybegins with the conversion of lysine to 2-amino-5-oxo-hexanoate (orα-aminoadipate semialdehyde) by a L-lysine-6-aminotransferase(classified as EC 2.6.1.36 and EC 1.4.1.18) as described in thebioproduction of C6 difunctional alkanes (enzymatic step 5a). In someembodiments, α-aminoadipate semialdehyde is converted to 5-aminopentanalby a decarboxylase (enzymatic step 7b). Examples of decarboxylaseinclude, but are not limited to, lysine decarboxylase, L-Ornithinecarboxylyase (EC 4.1.1.17, odc gene product and spec speF gene product),Aspartate 4-decarboxylase (EC 4.1.1.12), and glutamate decarboxylase(gadA (Escherichia coli K12), gadB (Escherichia coli K12), GAD1(Saccharomyces cerevisiae S288C), GAD2 and GAD 1 (Homo sapiens,Arabidopsis thaliana col). 5-aminopentanal can be converted to5-aminopentanol by an alcohol dehydrogenase or to 5-aminopentanoate byan aldehyde dehydrogenase.

In some embodiments, α-aminoadipate semialdehyde is converted toα-ketoadipate semialdehyde by a α-aminoadipate aminotransferase(enzymatic step 5b, EC 2.6.1.39, lysine degradation pathway). Theglutarate and the 1,5-pentanediol pathways involve a ketodecarboxylaseto convert α-ketoadipate semialdehyde to 5-oxopentanal (enzymatic step7e) and an alcohol dehydrogenase to convert 5-oxopentanal to1,5-pentanediol (enzymatic step 70 or an aldehyde dehydrogenase toconvert 2-oxopentanal to glutarate (enzymatic step 7g).

Although there are no known enzymes shown to catalyze thedecarboxylation of α-ketoadipate semialdehyde to 5-oxopentanal, a listof ketodecarboxylases was generated based on the following criteria: (i)demonstrated activity on a 2-ketocarboxylate, and (ii) availability ofprotein sequence information.

TABLE 10 4.1.1.71 2-ketoglutarate decarboxylase kgd from M. tuberculosisUniProt: O50463

4.1.1.1 2-ketoisovalerate decarboxylase kivD from L. lactis UniProt:Q684J7

4.1.1.43 Transaminated a.a. decarboxylase ARO10 from S. cerevisiaeUniProt: Q06408

4.1.1.7 Benzoylformate decarboxylase mdlC from P. putida crystalstructure available UniProt: P20906

4.1.1.75 2-ketoarginine decarboxylase aruI; P. aeruginosa NCBI AAG08362

4.1.1.82 Phosphonopyruvate decarboxylase fom2; Streptomyces wedmorensisUniProt: Q56190 NCBI AB016934

4.1.1.80 Pyruvate decarboxylase isozyme PDC6, PDC1; S. cerevisiae PDC1crystal structure available PDC1 UniProt: P06169 PDC6 UniProt: P26263

4.1.1.1 Pyruvate decarboxylase isozyme 2 PDC5, S. cerevisiae (also,PDC1, PDC6, Aro10, KivD) UniProt: P16467

4.1.1.74 Indolepyruvate decarboxylase ipdC; Pantoea agglomerans ipdC;Enterobacter cloacae P.a. UniProt P71323 E.c. UniProt P23234

4.1.1.74 Indolepyruvate decarboxylase ipdC; Pantoea agglomerans ipdC;Enterobacter cloacae P.a. UniProt P71323 E.c. UniProt Q47305

4.1.1.40 hydroxypyruvate decarboxylase gene unknown

Alcohol dehydrogenases (ADHs) (EC 1.1.1.1 and 1.1.1.2) catalyze thereversible reduction of ketones and aldehydes to alcohols with thereduction of NAD+ to NADH. In some embodiments, examples of alcoholdehydrogenases include, but are not limited to, adhA or adhB (from Z.mobilis), butanol dehydrogenase (from Clostridium acetobutylicum),propanediol oxidoreductase (from E. coli), ADHIV alcohol dehydrogenase(from Saccharomyces), and ADH6 (from S. cerevisiae). Aldehyde NAD(+)dehydrogenase activity and alcohol NAD(+) dehydrogenase activities canbe carried out by two different polypeptides, or carried out by a singlepolypeptide, such as a multi-functional aldehyde-alcohol dehydrogenase(EC 1.2.1.10) from E. coli (Goodlove et al. Gene 85:209-14, 1989;GenBank Accession No. M33504). Polypeptides having aldehydedehydrogenase (NAD(P)+) (EC 1.2.1.-) or aldehyde dehydrogenase (NAD(+))(EC 1.2.1.3) activity can be used in combination with an alcoholdehydrogenase to reduce the remaining carboxylic acid to an alcohol,yielding a diol. Nucleic acids encoding such polypeptides can beobtained from various species including, without limitation, S.cerevisiae.

In another embodiment, α-ketoadipate semialdehyde is converted toketoadipate by an aldehyde dehydrogenase including, but not limited to,acetaldehyde dehydrogenase (EC 1.2.1.4 encoded by aldB (Escherichia coliK12), ALD6 (Saccharomyces cerevisiae S288C), aldehyde dehydrogenase (EC1.2.1.3, ALD3 (Saccharomyces cerevisiae S288C), ALD2 (Saccharomycescerevisiae S288C), ALD4 (Saccharomyces cerevisiae S288C), ALD5(Saccharomyces cerevisiae S288C), ALDH2 (Homo sapiens), alkH(Pseudomonas oleovorans), Glycolaldehyde dehydrogenase (EC 1.2.1.21,aldA (Escherichia coli K12)), aldA (Escherichia coli K12)),L-lactaldehyde dehydrogenase (EC 1.2.1.22, MJ1411 (Methanocaldococcusjannaschii), alkH (Pseudomonas oleovorans), ALDH2 (Homo sapiens), ALD5(Saccharomyces cerevisiae S288C), ALD4 (Saccharomyces cerevisiae S288C),ALD2 (Saccharomyces cerevisiae S288C), and ALD3 (Saccharomycescerevisiae S288C)).

As illustrated in FIG. 7, enzymatic step 7h, 2-ketoadipate is thenconverted to glutarate semialdehyde by a keto-decarboxylase listed inTable 10 and then to 5-hydroxypentanoic acid by an alcoholdehydrogenase.

B. Engineered Pathways for the Bioproduction of C5 Difunctional Alkanesfrom Nitrogen-Containing Heterocyclic Ring

Aspects of the invention relate to the bioproduction of C5 difunctionalalkanes from nitrogen-containing heterocyclic ring (FIG. 8). One shouldappreciate that 2-aminoadipate semialdehyde can be generated from, forexample, L-pipecolate as discussed in the bioproduction of C6difunctional alkanes. 2-aminoadipate semialdehyde can then be convertedto C5 difunctional alkane as disclosed above. One should appreciate thatenzymes catalyzing enzymatic steps 5b may also catalyze enzymatic step7a; and enzymes catalyzing enzymatic step 5e may also catalyze enzymaticstep 7j.

III. Engineered Pathways for the Production of 6-Aminocaproic Acid andHexamethylenediamine from Lysine Via Carbon Extension

Aspects of the invention relate to the bioproduction of C6 difunctionalalkanes from lysine with C7 difunctional alkane intermediates (FIG. 9).

In some embodiments, the carbon chain of lysine is elongated to form a2,7-diaminoheptanoic acid. In some embodiments, the carbon chain oflysine is elongated following a pathway similar to the α-keto-elongationpathway. The α-keto-elongation pathway comprises three enzymes: anhomocitrate synthase, an homoaconitase and a homo-isocitratedehydrogenase. One would appreciate that the lysine biosynthesis pathwayIV provides enzymes for the elongation of 2-ketoglutarate toα-ketoadipate. For example, 2-ketoglutarate is converted to homocitrateby a homocitrate synthase (EC 2.3.3.14, LYS20 and LYS21 (Sacharomycescerevisiae), nifV (Klebsiella pneumoniae), hcs (Thermus thermophilus)).Homocitrate is then converted to cis-homoaconitate and then tohomoisocitrate by a homoaconitase (EC 4.2.1.36, LYS4 (Sacharomycescerevisiae), lysU, lysT (Thermus thermophilus)) and homoisocitrate isconverted to α-ketoadipate by a homo-isocitrate dehydrogenase (EC1.1.1.87, LYS12 (Sacharomyces cerevisiae), hicdh (Thermusthermophilus)).

In an engineered pathway for the bioproduction of hexamethylenediamine,2,7-diaminoheptanoic acid is converted to hexamethylenediamine in asingle enzymatic step catalyzed by a decarboxylase. In a preferredembodiment, the decarboxylase is lysine decarboxylase. Lysinedecarboxylases (EC 4.1.1.18) isolated from Escherichia coli havinglysine decarboxylase activity are the cadA gene product and the ldc geneproduct. Other decarboxylases capable of catalyzing the substrate toproduct reaction are listed in Table 9.

In some embodiments, a 2,7-diaminoheptanoic acid is converted to7-amino-2-oxoheptanoic acid by an aminotransferase enzyme (EC 2.6.1.x)or a dehydrogenase (EC 1.4.1.x or EC 1.4.3.x). Preferred enzymes arelisted in Table 7 and may be engineered to catalyze the desiredsubstrate to product reaction.

In an alternative embodiment, lysine is first converted to6-amino-2-oxohexanoic acid by an aminotransferase enzyme (EC 2.6.1.x) ora dehydrogenase (EC 1.4.1.x or EC 1.4.3.x). Preferred enzymes are listedin Table 7 and may be engineered to catalyze the desired substrate toproduct reaction. In a subsequent step, 6-amino-2-oxohexanoic acid issubjected to a carbon elongation enzymatic step to produce7-amino-2-oxoheptanoic acid.

In a subsequent step, 7-amino-2-oxoheptanoic acid is converted to6-aminohexanal by a decarboxylase. Preferred decarboxylases are lysinedecarboxylases (EC 4.1.1.18). However, in some embodiments, the7-amino-2-oxoheptanoic acid to 6-aminohexanal conversion is catalyzed byan enzyme listed in Table 9. 6-aminohexanal is subsequently converted to6-aminocaproic acid by an aldehyde dehydrogenase (EC 1.2.1.3) oraldehyde oxidase (EC 1.2.3.1, AAO2 (Arabidopsis thaliana col), AAO1(Arabidopsis thaliana col), AOX1 (Homo sapiens)).

Yet, in another embodiment, 2,7-diaminoheptanoic acid is converted to6-aminohexamide by a monooxygenase. Preferred monooxygenase is aL-lysine monooxygenase (EC 1.13.12.12, davB in Pseudomonas fluorescens)that catalyses the conversion of L-lysine to 5-aminopentamide in thelysine degradation IV pathway. In a subsequent step, 6-aminohexamide isconverted to 6-aminocaproic acid by an amidase. In one embodiment, theamidase is a δ-aminovaleramidase (EC 3.5.1.30, davA (Pseudomonasputida)) that catalyses the conversion of 5-aminopentanamide to5-aminopentanoate in the lysine degradation IV pathway.

III. Culture Conditions and Screening Techniques

Microorganisms may be cultivated continuously or discontinuously in abatch process (batch cultivation) or in a fed-batch process (feedprocess) or repeated fed-batch process (repetitive feed process) for thepurposes of difunctional alkanes.

The culture medium to be used must satisfy in a suitable manner therequirements of the respective strains. Descriptions of culture mediafor various microorganisms are contained in the handbook “Manual ofMethods for General Bacteriology” of the American Society forBacteriology (Washington D.C., USA, 1981). Media must contain suitablecarbon sources such as monosaccharides (e.g. glucose and fructose),oligosaccharides (e.g. sucrose, lactose), polysaccharides (e.g. starchand cellulose), oils and fats or mixture thereof. Media must contain anitrogen source such as organic nitrogen-containing compounds such aspeptones, yeast extract, meat extract, malt extract, corn steep liquor,soy bean flour and urea, or inorganic compounds such as ammoniumsulfate, ammonium chloride, ammonium phosphate, ammonium carbonate andammonium nitrate. The nitrogen sources may be used individually or as amixture.

In addition to the carbon sources and nitrogen sources, media mustcontain suitable minerals, salts, cofactors, buffers and othercomponents, known to those skilled in the art, suitable for growth ofthe culture and promotion of the enzymatic pathways for C5 and C6difunctional alkanes.

Typically cells are grown at a temperature in the range of 20° C. toabout 45° C. and preferably 25° C. to 40° C. in an appropriate medium.Suitable growth media includes common commercially available media suchas Luria Bertani (LB) broth, Yeast medium (YM) or any synthetic ordefined media. Suitable pH ranges are between pH 5.0 to pH 9.0. In orderto regulate the pH of the culture, basic compounds such as sodiumhydroxide, potassium hydroxide, ammonia or ammonia water, or acidiccompounds such as phosphoric acid or sulfuric acid are used asappropriate. Culture may be performed under aerobic or anaerobicconditions.

Screening Techniques

In accordance with the methods described herein, reaction mixtures forpathway development may be carried out in any vessel that permits cellgrowth and/or incubation. For example, a reaction mixture may be abioreactor, a cell culture flask or plate, a multiwell plate (e.g., a96, 384, 1056 well microtiter plates, etc.), a culture flask, afermentor, or other vessel for cell growth or incubation.

Screening may be carried out by detection of expression of a selectablemarker, which, in some genetic circumstances, allows cells expressingthe marker to survive while other cells die (or vice versa). Efficientscreening techniques are needed to provide efficient development ofnovel pathways using the methods described herein. Preferably, suitablescreening techniques for compounds produced by the enzymatic pathwaysallow for a rapid and sensitive screen for the properties of interest.Visual (colorimetric) assays are optimal in this regard, and are easilyapplied for compounds with suitable light absorption properties. Moresophisticated screening technologies include, for instance,high-throughput HPLC-MS analysis, SPME (Solid Phase Microextraction) andGC-MS (Gas chromatography-mass spectrometry) (see Handbook of analyticalderivatization reaction, D. R. Knapp; John Wiley & Sons, 1979). In someinstance, screening robots are connected to HPLC-MS systems forautomated injection and rapid sample analysis. These techniques allowfor high-throughput detection and quantification of virtually anydesired compound.

Biologically produced products of interest may be isolated from thefermentation medium or cell extract using methods known in the art. Forexample, solids or cell debris may be removed by centrifugation,filtration, decantation and the like. Bioproducts of interest may beisolated by distillation, liquid-liquid extraction, membraneevaporation, adsorption, or using any methods known in the art.

In some embodiments, identification of the product of interest may beperformed using an HPLC. For example, the standard samples are preparedwith known amounts of the organic product in the medium (e.g. adipicacid, amino caproic acid). The retention time of the adipic acidproduced can then be compared to that of the authentic standard. In someembodiments, identification of the product of interest may be performedusing a GC-MS. The resolved samples are then analyzed by a massselective detector and compared to previous mass spectra and retentiontime of authentic standards.

In some embodiments, cellular extracts may be screened for enzymeactivity. For example, oxohexanoate dehydrogenase activity may bedetected by measuring the rate of increase of absorbance at 340 nm asdescribed in Donoghue and Trudgill (Eur. J. Biochem., 1975, 60:1-7).

The practice of the present methods will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, engineering, robotics, optics, computer software andintegration. The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences. Such techniques are explained fully in the literature. See,for example, Molecular Cloning A Laboratory Manual, 2^(nd) Ed., ed. bySambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press:1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985);Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S.Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J.Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J.Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R.Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); the treatise,Methods In Enzymology (Academic Press, Inc., N.Y.); Gene TransferVectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987,Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155(Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987); Handbook OfExperimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,eds., 1986); Manipulating the Mouse Embryo, (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1986); Lakowicz, J. R.Principles of Fluorescence Spectroscopy, New York:Plenum Press (1983),and Lakowicz, J. R. Emerging Applications of Fluorescence Spectroscopyto Cellular Imaging: Lifetime Imaging, Metal-ligand Probes, Multi-photonExcitation and Light Quenching, Scanning Microsc. Suppl. VOL. 10 (1996)pages 213-24, for fluorescent techniques, Optics Guide 5 Melles Griot®Irvine Calif. for general optical methods, Optical Waveguide Theory,Snyder & Love, published by Chapman & Hall, and Fiber Optics Devices andSystems by Peter Cheo, published by Prentice-Hall for fiber optic theoryand materials.

EQUIVALENTS

The present invention provides among other things compositions andmethods for metabolic engineering. While specific embodiments of thesubject invention have been discussed, the above specification isillustrative and not restrictive. Many variations of the invention willbecome apparent to those skilled in the art upon review of thisspecification. The full scope of the invention should be determined byreference to the claims, along with their full scope of equivalents, andthe specification, along with such variations.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein, including those itemslisted below, are hereby incorporated by reference in their entirety asif each individual publication or patent was specifically andindividually indicated to be incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

We claim:
 1. A recombinant microorganism producing 6-aminocaproic acidfrom lysine, the recombinant microorganism comprising a firstrecombinant nucleic acid encoding a first polypeptide that catalyzes asubstrate to product conversion of beta-lysine to3,6-diaminohexanoyl-CoA, wherein the first recombinant nucleic acidmolecule is heterologous to the recombinant microorganism.
 2. Arecombinant microorganism producing 6-aminocaproic acid from lysine, therecombinant microorganism comprising at least one recombinant nucleicacid encoding a polypeptide that catalyzes a substrate to productconversion of 6-amino-3-hydroxyhexanoyl-CoA to 6-aminohex-2-enoyl-CoA.3. The recombinant microorganism of claim 1, further comprising a secondrecombinant nucleic acid encoding a second polypeptide that catalyzes asubstrate to product conversion of 6-aminohex-2-enoic acid to6-aminocaproic acid.
 4. The recombinant microorganism of claim 1,wherein the first recombinant nucleic acid molecule comprises anengineered nucleic acid having less than 95% identity with a naturalnucleic acid.
 5. The recombinant microorganism of claim 3, wherein thepolypeptides that catalyze substrate to product conversion do not existin a natural biological system.
 6. The recombinant microorganism ofclaim 1, further comprising a second recombinant nucleic acid encoding asecond polypeptide that catalyzes conversion of 6-aminohexanoyl-CoA to6-aminocaproic acid.